Fgf21 c-terminal peptide optimization

ABSTRACT

Disclosed herein are modified C-terminal fragments of FGF21 optimized for binding to Klotho β or antagonizing FGF21 activity. FGF21 peptides modified to comprise modifications to the C-terminal amino acid sequence are disclosed that have enhanced activity at the FGF21 receptor. Additionally, conjugates formed between the optimized FGF21 peptide fragments and insulin like peptides or nuclear hormone receptor ligands are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/323,003 filed on Apr. 15, 2016, the disclosure of which is herebyexpressly incorporated by reference in its entirety.

INCORPORATION BY REFERENCES OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 165 kilobytes ACII (Text) file named“PCTKlotho_ST25.txt,” created on Apr. 13, 2017.

BACKGROUND

Fibroblast growth factor 21 (FGF21) is a recently identified circulatingprotein that regulates insulin sensitivity along with lipid and energymetabolism. FGF21 belongs to a subfamily of Fibroblast Growth Factors(FGFs) that includes FGF19 (SEQ ID NO: 170), FGF21 (SEQ ID NO: 171), andFGF23 (SEQ ID NO: 172). FGF is expressed with a 28 amino acid signalpeptide that is subsequently cleaved to produce the mature protein (SEQID NO: 173) FGF21 is an atypical FGF in that it is heparin independentand functions as a hormone in the regulation of glucose, lipid, andenergy metabolism.

FGF21 is highly expressed in liver and pancreas and is the only memberof the FGF family to be primarily expressed in liver. Transgenic miceoverexpressing FGF21 exhibit metabolic phenotypes of slow growth rate,low plasma glucose and triglyceride levels, and an absence ofage-associated type 2 diabetes, islet hyperplasia, and obesity.

Pharmacological administration of recombinant FGF21 protein in obesediabetic rodents markedly improved hyperglycemia, lowered elevatedtriglycerides (TGs), and reduced body weight. Similarly, administrationto obese diabetic rhesus monkeys results in normalized levels of plasmaglucose, reduced triglyceride and cholesterol levels, and improvedglucose tolerance and insulin sensitivity. FGF21 functions to reducebody weight and body fat by increasing energy expenditure, physicalactivity, and metabolic rate. Experimental research (see Gaich et al.,Cell Metab. Vol. 18(3):333-40 (September 2013)) provides support for thepharmacological administration of FGF21 for the treatment of type 2diabetes, obesity, dyslipidemia, and other metabolic conditions ordisorders in humans.

At a molecular level, FGF21 interacts with the FGF receptor only intissues expressing the cofactor Klotho β. The Klotho β-dependent tissuespecificity is consistent with the predominant effects of FGF21occurring in liver and adipose tissue. The terminal residues of FGF21are vital for effective biochemical signaling and their truncationdramatically affects the biochemical activity of the molecule. While itis evident that the C-termini of the hormonal FGFs are important forfacilitating the interaction with their respective co-receptor partnersfor the formation of an active receptor complex, the detailed molecularrequirements for the association are unknown.

The beneficial pharmacology observed in preclinical models indicatedthat FGF21 and its analogs or mimetics hold promise as innovativetherapeutics for treating metabolic disorders. However, analogs of FGF21having greater potency at the FGF receptor is desirable to enhance theefficacy of FGF21 mediated therapies

SUMMARY

In accordance with one embodiment of the present disclosure a method ofidentifying an optimized FGF21 analog is provided. In one embodiment themethod of identifying an optimized FGF21 analog is based on analyzingthe C-terminal 25 amino acid peptide fragment of FGF21 (SEQ ID NO: 166)for determining the structure-activity relationship for protein FGF21.Applicants have found that in terms of its ability to antagonize FGF21activity, the peptide of SEQ ID NO: 166, and derivatives thereof, arealso predictive of FGF receptor activity of the whole FGF proteincomprising a derivative peptide of SEQ ID NO: 166. Accordingly, in oneembodiment the method of identifying an optimized FGF21 analog comprisesthe steps of modifying a peptide comprising the sequencePPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID NO: 166), analyzing the modifiedpeptide's ability to bind to Klotho β or antagonize FGF21 activity in anin vitro assay (e.g., the assay of Example 1) and identifying modifiedpeptides that have an enhanced activity relative to the native peptide(SEQ ID NO: 166). The identified modified peptides can then beincorporated into a full length protein, such as the FGF21 protein orother bioactive proteins, using standard means such as biosynthesis orsemi-synthesis.

In accordance with one embodiment peptides based on the C-terminal 25amino acids of native FGF21 and FGF19 are provided that have an enhancedability to bind Klotho β and function as antagonist of FGF21 activityrelative to the native sequence. In accordance with one embodiment a 25amino acid peptide selected from the group consisting of

LETDSMDPFGLVTGLEAVRSPSFEA (SEQ ID NO: 188),

PPDVGSSDPLSMVGPSQGRSPSYAA (SEQ ID NO: 191),

PPDVGSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 180),

PLETDSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 179),

PDVGSMDPFGLVTGLEAVRSPSYAA (SEQ ID NO: 234),

PPDVG SMDPF GLVGR SQGRS PSFEA (SEQ ID NO: 237),

PPDVF SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 238),

PPDVL SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 239),

PPDVS SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 240), and

PPDVG SSDPF GLVGP SQGRS PSFEA (SEQ ID NO: 241) is provided. Each ofthese peptides have been found to be more potent in antagonizing thenative FGF21's in vitro signaling than the corresponding native FGF21C-terminal 25 amino acid fragment.

Substituting any of the novel sequences of SEQ ID NOs: 179, 180, 188,191, 234, 237, 238, 239, 240 or 241 for the native C-terminal 25 aminoacids of FGF21 produces an FGF analog that has higher potency at the FGFreceptor than native FGF21. Furthermore, applicants have discoveredadditional modifications to the native FGF21 sequence that also enhancethe peptide's activity at its receptor. In accordance with oneembodiment an agonist analog of FGF21 is provided having enhancedpotency at the FGF receptor, where said analog comprises at least oneamino acid modification selected from the group consisting of aminoacids positions 159, 160, 162, 164, 165,166, 168, 169, 176, 177, and179, relative to SEQ ID NO: 173. In one embodiment the amino acidmodification comprises an amino acid substitution with a non-naturalamino acid. In one embodiment the non-natural amino acid substitution isa substitution of an amino acid with an amino acid in the D-stereoconfiguration, and optionally the corresponding D-stereoisomer of thenative amino acid at that position. In one embodiment an analog of FGF21is provided having enhanced potency relative to the native FGF21sequence of SEQ ID NO: 167, where said analog comprises 1-6, 1-4, or 1-2amino acid modifications at amino acid positions selected from positions159, 160, 162, 164, 165,166, 168, 169, 176, 177, and 179, relative toSEQ ID NO: 167. In one embodiment an analog of FGF21 is provided havingenhanced potency, where said analog comprises 1-2 amino acidmodifications selected from the group consisting of amino acidspositions 164, 165 and 168, relative to SEQ ID NO: 167.

In accordance with one embodiment an FGF21 analog is provided havingenhanced potency at the FGF receptor, wherein the C-terminal amino acidis substituted with a small aliphatic amino acid, optionally whereinsaid small aliphatic amino acid has a C₁-C₄ side chain. In accordancewith one embodiment the C-terminal amino acid is substituted with anamino acid selected from the group consisting of glycine, alanine,valine, isoleucine and leucine. In one embodiment the C-terminal aminoacid is substituted with alanine. In a further embodiment an FGF21analog is provided having enhanced potency at the FGF receptor whereinthe C-terminal 25 amino acids of FGF21 are substituted with the nativeC-terminal 25 amino acids of FGF19, further modified by substituting theC-terminal amino acid of the FGF21 analog with alanine.

In a further embodiment novel conjugates comprising a modifiedC-terminal 25 amino acid peptide of FGF21 are provided. In oneembodiment the conjugate comprises an insulin agonist peptide or an FGF1peptide covalently linked to a modified peptide of SEQ ID NO: 166,wherein the modified peptide differs from SEQ ID NO: 166 by one or moreamino acid modifications at positions selected from the group consistingof 3, 4, 6, 8, 9, 10, 12, 13, 20, 21 and 23 of SEQ ID NO: 166. In oneembodiment the conjugate comprises a modified peptide of SEQ ID NO: 166wherein the modified peptide differs from SEQ ID NO: 166 by 1, 2, 3, 4or 5 amino acid substitution at amino acid positions selected from thegroup consisting of 3, 4, 6, 8, 9, 10, 12, 13, 20, 21 and 23. In oneembodiment the conjugate comprises a modified peptide of SEQ ID NO: 166wherein the modified peptide differs from SEQ ID NO: 166 by 1 or 2 aminoacid substitution at amino acid positions selected from the groupconsisting of 8, 9 and 12. In accordance with one embodiment theconjugate comprises a 25 amino acid peptide selected from the groupconsisting of (SEQ ID NO: 188), (SEQ ID NO: 191), (SEQ ID NO: 180), (SEQID NO: 179), (SEQ ID NO: 234), (SEQ ID NO: 237), (SEQ ID NO: 238), (SEQID NO: 239), (SEQ ID NO: 240), and (SEQ ID NO: 241).

In accordance with one embodiment conjugates of the present disclosurecan be represented by the following formula:

Q-L-Y

wherein Q is an insulin peptide, a glucagon peptide, FGF1, FGF2, ornuclear hormone, Y is a peptide comprising the sequence of SEQ ID NO:166 or a modified peptide that differs from SEQ ID NO: 166 by 1, 2, 3, 4or 5 amino acid substitution at amino acid positions selected from thegroup consisting of 3, 4, 6, 8, 9, 10, 12, 13, 20, 21 and 23 of SEQ IDNO: 166, and L is a linking group or a bond. In accordance with oneembodiment Y is a 25 amino acid peptide selected from the groupconsisting of (SEQ ID NO: 188), (SEQ ID NO: 191), (SEQ ID NO: 180), (SEQID NO: 179), (SEQ ID NO: 234), (SEQ ID NO: 237), (SEQ ID NO: 238), (SEQID NO: 239), (SEQ ID NO: 240), and (SEQ ID NO: 241). In one embodiment Qis an insulin peptide, or nuclear hormone. The insulin peptide componentof the conjugate can be native insulin or any known insulin analog thathas activity at the insulin receptor including, for example, any insulinpeptide disclosed in published international applications WO96/34882, WO2010/080607, WO 2010/080609, WO 2011/159882, WO/2011/159895 and U.S.Pat. No. 6,630,348, the disclosures of which are incorporated herein byreference. In embodiments where Q is an NHR ligand, the ligand is whollyor partly non-peptidic and acts at a nuclear hormone receptor with anactivity in accordance with any of the teachings set forth herein. Insome embodiments the NHR ligand is an agonist that, in its unboundstate, has an EC50 or IC50 of about 1 mM or less, or 100 μM or less, or10 μM or less, or 1 μM or less. In accordance with one embodiment theNHR ligand component of the conjugate can be a ligand that activates thethyroid hormone receptor or activates the peroxisomeproliferator-activated receptors (PPAR) when in an unbound state.

In other aspects of the present disclosure, methods are provided foradministering a therapeutically effective amount of a Q-L-Y conjugate orFGF21-based analog described herein for treating a disease or medicalcondition in a patient. In some embodiments, the disease or medicalcondition is selected from the group consisting of metabolic syndrome,diabetes, obesity, liver steatosis, and chronic cardiovascular disease.In one embodiment the FGF21-based analogs are administered to a patientto treat metabolic syndrome and lipid abnormalities of the liver,including for example non-alcoholic steatohepatitis (NASH).

Also encompassed by the present disclosure are pharmaceuticalcompositions comprising the conjugates or FGF21-based analogs disclosedherein and a pharmaceutically acceptable carrier. In accordance with oneembodiment a pharmaceutical composition is provided comprising any ofthe conjugates or FGF21-based analogs disclosed herein preferably at apurity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%, and a pharmaceutically acceptable diluent, carrier or excipient.Such compositions may contain a conjugate or FGF21-based analog asdisclosed herein at a concentration of at least 0.5 mg/ml, 1 mg/ml, 2mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24mg/ml, 25 mg/ml or higher. In one embodiment the pharmaceuticalcompositions comprise aqueous solutions that are sterilized andoptionally stored within various package containers. In otherembodiments the pharmaceutical compositions comprise a lyophilizedpowder. The pharmaceutical compositions can be further packaged as partof a kit that includes a disposable device for administering thecomposition to a patient. The containers or kits may be labeled forstorage at ambient room temperature or at refrigerated temperature.

In accordance with one embodiment an improved method of regulating bloodglucose levels in insulin dependent patients is provided. The methodcomprises the steps of administering to a patient an FGF21-based peptideanalog or conjugate as disclosed herein in an amount therapeuticallyeffective for the control of diabetes. In accordance with one embodimenta method of reducing weight or preventing weight gain is providedwherein the method comprises administering a conjugate or FGF21-basedanalog as disclosed herein to a patient in need of such therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a graph demonstrating C-terminal peptides of FGF21 (aminoacids 153-181) and FGF19 (amino acids 165-194) exhibit antagonism toFGF21 activity (10 nM stimulation) in 293 HEK hKLB cells equivalent to amuch larger fragment of FGF21 (amino acids 18-181).

FIG. 2 is a graph comparing the ability of varying lengths of C-terminalpeptides of FGF21 to antagonize to FGF21 activity (10 nM stimulation) in293 HEK hKLB cells. The results demonstrate a terminal fragment ofgreater than 20 amino acids is required for antagonism of FGF21activity.

FIGS. 3A & 3B are graphs demonstrating that the C-terminal 25 amino acidfragment of FGF21 (FIG. 3A) or FGF19 (FIG. 3B) is sufficient forantagonism of FGF21 activity. Also provided is the activity of variousalanine-mutated peptides of FGF21 157-181 as antagonists to FGF21activity (FIG. 3A).

FIG. 4 is a graph presenting data for FGF21 analogs with alaninemutations at positions 164 and 171 compared to native FGF21 activity.Alanine substitution at position 164 was found to significantly impactFGF21 activity.

FIG. 5 provides the Far-UV CD spectra comparing native FGF21 and theAla164 site-specific analog demonstrating the loss of antagonism withthe Ala164 is not associated with changes in the secondary structure forthe peptide.

FIG. 6 shows the sequence alignment of the C-termini of FGF21 and FGF19.

FIGS. 7A-7C provided data regarding the bioactivity of FGF21 157-181 orFGF19 169-194 alanine scan mutation analogs. FIGS. 7A & 7B present bargraphs plotting the respective bioactivity of the FGF21 157-181 or FGF19169-194 alanine mutation analogs. The residue positions are denoted withregard to the FGF19 sequence. FIG. 7A presents the fold change in thepotencies of the set of analogs achieving complete antagonistic responsein comparison to its respective native FGF21 or FGF19 peptide. FIG. 7Bpresents % maximal activities of the set of analogs with less than 95%maximal activity with respect to their native FGF21 or FGF19 peptide.The vertical dotted line represents the activities of the nativepeptides. Surprisingly, a significant difference in bioactivity wasachieved in FGF21 and FGF19 analogs having Ala in the C-terminalposition. FIG. 7C is a graph demonstrating the superior bioactivity ofFGF19 169-194 K194A peptide to antagonize FGF21 activity in Hep3B cellsrelative to the native FGF21 18-181, FGF21 157-181 and FGF19 169-194sequences, thus confirming the unexpected activity associated with theK194A substitution.

FIGS. 8A & 8B. are graphs representing the consequences of substitutionsat the terminal position of the antagonist peptide. FIG. 8A graphs thedata produced from a mutational analysis at the terminal position ofFGF19 169-194 peptide and its effect on the antagonistic activity, IC-50values provided in [nM]. K194A [10.5] (squares); K194S [22.3] (circles);K194L [17.3] (triangles); K194E [87.9] (inverted triangles). FIG. 8Bgraphs the data produced from an analysis of the presence or absence ofa lysine substitution at terminal position in FGF21 and FGF19 C-terminalpeptides. FGF21 157-181 [0.04] (squares); FGF21 157-181 S181K (circles);FGF19 169-194 [0.05] (triangles); FGF19 169-194 K194A [0.0005] (invertedtriangles), IC-50 values in [μM].

FIGS. 9A & 9B. are graphs demonstrating the translatability between theeffects of alanine substitution seen in the antagonist peptides andtheir corresponding full length agonist analogs. Selected alaninemutations from the antagonist peptides were incorporated into theircorresponding full length FGF21 or FGF19 protein sequences and testedfor their ability to activate Erk1/2 phosphorylation in 293T HEK hKLBcells. FIG. 9A shows the activity for FGF21 [4.5, 100] (squares); FGF21D164A [inactive, 6] (circles); FGF21 P171A [4.5, 100] (triangles). FIG.9B shows the activity for FGF21 [3.3, 100] (squares); FGF19 [147.9, 27](circles); FGF19 K194A [8.1, 31] (triangles); FGF21-19 K194A [0.5, 84](inverted triangles). EC-50 and % maximal activity values are providedin [nM, %].

FIGS. 10A & 10 B are graphs demonstrating the FGF21 and FGF19 peptidesare functional in mouse as well as human cells. FGF21 and FGF19 peptideswere acetylated at the N-terminus to help increase stability for use invivo. Acetylated FGF21 and FGF19 peptides retain their antagonisticactivity at both human (FIG. 10A) and mouse (FIG. 10B) cells thatoverexpressing human and mouse Klotho β (KLB), respectively.

FIGS. 11A & 11B are graphs demonstrating that modification of FGF21 byreplacing the native C-terminal 25 amino acids with the FGF19 A26antagonistic peptide significantly increases agonism at both human (FIG.11A) and mouse (FIG. 11B) KLB. This fusion, called 0268-1A, has enhancedpotency (1.82±0.37 & 2.15±0.54) compared to both FGF21 (8.42±4.1 &3.96±1.51) and FGF19 (65.78±58.19 & 25.67±21.32) in cells thatoverexpressed both human and mouse KLB.

FIG. 12 is a graph demonstrating modification of the N-Terminus of FGF21analog 0278-1A by amino acid substitutions A31C, G43C, L98D, L100K,N121D, and D127K (position numbering based on the native FGF21 sequence)to generate analog V2-0278-1A, increases potency relative to 0278-1A.V2-0278-1A displays ˜2-3× higher potency (0.35±0.12 nM) versus 0278(1.13±0.28) in cells overexpressing human KLB.

FIGS. 13A-13C provide data relating to the in vivo administration of theFGF21 analog of SEQ ID NO: 193 (analog 0361) administered at a dosage of0.3 mg/kg or 1.0 mg/kg. FIGS. 13A and 13B demonstrate the superiorchange in body weight obtained with the FGF21 analog relative to thecontrol and administered native FGF21, as measured by total body weight(FIG. 13A) or by percent change in body weight (FIG. 13B). FIG. 13Cdemonstrates those mice receiving the FGF21 analog had a greaterreduction in total food intake.

DETAILED DESCRIPTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

As used herein the term “amino acid” encompasses any molecule containingboth amino and carboxyl functional groups, wherein the amino andcarboxylate groups are attached to the same carbon (the alpha carbon).The alpha carbon optionally may have one or two further organicsubstituents. For the purposes of the present disclosure designation ofan amino acid without specifying its stereochemistry is intended toencompass either the L or D form of the amino acid, or a racemicmixture.

As used herein the term “hydroxyl acid” refers to amino acids that havebeen modified to replace the alpha carbon amino group with a hydroxylgroup.

As used herein the term “non-coded amino acid” encompasses any aminoacid that is not an L-isomer of any of the following 20 amino acids:Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, Tyr.

As used herein a general reference to a peptide is intended to encompasspeptides that have modified amino and carboxy termini. For example, anamino acid sequence designating the standard amino acids is intended toencompass standard amino acids at the N- and C-terminus as well as acorresponding hydroxyl acid or acetylated amino acid at the N-terminusand/or a corresponding C-terminal amino acid modified to comprise anamide group in place of the terminal carboxylic acid.

As used herein an “acylated” amino acid is an amino acid comprising anacyl group which is non-native to a naturally-occurring amino acid,regardless by the means by which it is produced. Exemplary methods ofproducing acylated amino acids and acylated peptides are known in theart and include acylating an amino acid before inclusion in the peptideor peptide synthesis followed by chemical acylation of the peptide. Insome embodiments, the acyl group causes the peptide to have one or moreof (i) a prolonged half-life in circulation, (ii) a delayed onset ofaction, (iii) an extended duration of action, (iv) an improvedresistance to proteases, such as DPP-IV, and (v) increased potency atthe IGF and/or insulin peptide receptors.

As used herein, an “alkylated” amino acid is an amino acid comprising analkyl group which is non-native to a naturally-occurring amino acid,regardless of the means by which it is produced. Exemplary methods ofproducing alkylated amino acids and alkylated peptides are known in theart and including alkylating an amino acid before inclusion in thepeptide or peptide synthesis followed by chemical alkylation of thepeptide. Without being held to any particular theory, it is believedthat alkylation of peptides will achieve similar, if not the same,effects as acylation of the peptides, e.g., a prolonged half-life incirculation, a delayed onset of action, an extended duration of action,an improved resistance to proteases, such as DPP-IV, and increasedpotency at the IGF and/or insulin receptors.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

As used herein, the term “hydrophilic moiety” refers to any compoundthat is readily water-soluble or readily absorbs water, and which aretolerated in vivo by mammalian species without toxic effects (i.e. arebiocompatible). Examples of hydrophilic moieties include polyethyleneglycol (PEG), polylactic acid, polyglycolic acid, apolylactic-polyglycolic acid copolymer, polyvinyl alcohol,polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline,polyhydroxyethyl methacrylate, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatised cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose and co-polymersthereof, as well as natural polymers including, for example, albumin,heparin and dextran.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating diabetes” willrefer in general to maintaining glucose blood levels near normal levelsand may include increasing or decreasing blood glucose levels dependingon a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a compound or conjugate refers to a nontoxic but sufficientamount of the compound or conjugate to provide the desired effect. Theamount that is “effective” will vary from subject to subject, dependingon the age and general condition of the individual, mode ofadministration, and the like. Thus, it is not always possible to specifyan exact “effective amount.” However, an appropriate “effective” amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route such as intranasal, inhalation, subcutaneous,intramuscular, intraspinal, or intravenous.

Throughout the application, all references to a particular amino acidposition in an insulin analog by letter and number (e.g. position A5)refer to the amino acid at that position of either the A chain (e.g.position A5) or the B chain (e.g. position B5) in the respective nativehuman insulin A chain (SEQ ID NO: 1) or B chain (SEQ ID NO: 2), or thecorresponding amino acid position in any analogs thereof. For example, areference herein to “position B28” absent any further elaboration wouldmean the corresponding position B27 of the B chain of an insulin analogin which the first amino acid of SEQ ID NO: 2 has been deleted.Similarly, amino acids added to the N-terminus of the native B chain arenumbered starting with B0, followed by numbers of increasing negativevalue (e.g., B-1, B-2 . . . ) as amino acids are added to theN-terminus. Alternatively, any reference to an amino acid position inthe linking moiety of a single chain analog, is made in reference to thenative C chain of IGF 1 (SEQ ID NO: 17). For example, position 9 of thenative C chain (or the “position C9”) has an alanine residue.

As used herein the term “native insulin peptide” is intended todesignate the 51 amino acid heteroduplex comprising the A chain of SEQID NO: 1 and the B chain of SEQ ID NO: 2, as well as single-chaininsulin analogs that comprise SEQ ID NOS: 1 and 2. The term “insulinpeptide” as used herein, absent further descriptive language is intendedto encompass the 51 amino acid heteroduplex comprising the A chain ofSEQ ID NO: 1 and the B chain of SEQ ID NO: 2, as well as single-chaininsulin analogs thereof (including for example those disclosed inpublished international application WO96/34882 and U.S. Pat. No.6,630,348, the disclosures of which are incorporated herein byreference), including heteroduplexes and single-chain analogs thatcomprise modified analogs of the native A chain and/or B chain andderivatives thereof. Such modified analogs include modification of theamino acid at position A19, B16 or B25 to a 4-amino phenylalanine or oneor more amino acid substitutions at positions selected from A5, A8, A9,A10, A12, A14, A15, A17, A18, A21, B1, B2, B3, B4, B5, B9, B10, B13,B14, B17, B20, B21, B22, B23, B26, B27, B28, B29 and B30 or deletions ofany or all of positions B1-4 and B26-30. Insulin peptides as definedherein can also be analogs derived from a naturally occurring insulin byinsertion or substitution of a non-peptide moiety, e.g. a retroinversofragment, or incorporation of non-peptide bonds such as an azapeptidebond (CO substituted by NH) or pseudo-peptide bond (e.g. NH substitutedwith CH₂) or an ester bond (e.g., a depsipeptide, wherein one or more ofthe amide (—CONHR—) bonds are replaced by ester (COOR) bonds).

As used herein the term “insulin-like peptide” is intended to designateinsulin peptides and related peptides that share the common structuralelement of having six cysteine residues that form the three disulfidecross-links similar to native insulin. Such related peptides includeinsulin like growth factors (e.g., IGF I and IGF II), insulin likepeptides (e.g., insulin like peptides 3, 4, 5 and 6) and relaxins (e.g.,relaxin-1, 2 and 3).

As used herein, the term “single-chain insulin analog” encompasses agroup of structurally-related proteins wherein insulin or IGF A and Bchains, or analogs or derivatives thereof, are covalently linked to oneanother to form a linear polypeptide chain. As disclosed herein thesingle-chain insulin analog comprises the covalent linkage of thecarboxy terminus of the B chain to the amino terminus of the A chain viaa linking moiety.

As used herein the term “insulin A chain”, absent further descriptivelanguage is intended to encompass the 21 amino acid sequence of SEQ IDNO: 1 as well as functional analogs and derivatives thereof, includinginsulin analogs known to those skilled in the art, includingmodification of the sequence of SEQ ID NO: 1 by one or more amino acidinsertions, deletions or substitutions at positions selected from A4,A5, A8, A9, A10, A12, A14, A15, A17, A18, A21.

As used herein the term “insulin B chain”, absent further descriptivelanguage is intended to encompass the 30 amino acid sequence of SEQ IDNO: 2, as well as modified functional analogs of the native B chain,including one or more amino acid insertions, deletions or substitutionsat positions selected from B1, B2, B3, B4, B5, B9, B10, B13, B14, B17,B20, B21, B22, B23, B25, B26, B27, B28, B29 and B30 or deletions of anyor all of positions B1-4 and B26-30.

The term “identity” as used herein relates to the similarity between twoor more sequences. Identity is measured by dividing the number ofidentical residues by the total number of residues and multiplying theproduct by 100 to achieve a percentage. Thus, two copies of exactly thesame sequence have 100% identity, whereas two sequences that have aminoacid deletions, additions, or substitutions relative to one another havea lower degree of identity. Those skilled in the art will recognize thatseveral computer programs, such as those that employ algorithms such asBLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol.Biol. 215:403-410) are available for determining sequence identity.

As used herein, the term “selectivity” of a molecule for a firstreceptor relative to a second receptor refers to the following ratio:EC₅₀ of the molecule at the second receptor divided by the EC₅₀ of themolecule at the first receptor. For example, a molecule that has an EC₅₀of 1 nM at a first receptor and an EC₅₀ of 100 nM at a second receptorhas 100-fold selectivity for the first receptor relative to the secondreceptor.

As used herein an amino acid “modification” refers to a substitution ofan amino acid, or the derivation of an amino acid by the addition and/orremoval of chemical groups to/from the amino acid, and includessubstitution with any of the 20 amino acids commonly found in humanproteins, as well as atypical or non-naturally occurring amino acids.Commercial sources of atypical amino acids include Sigma-Aldrich(Milwaukee, Wis.), ChemPep Inc. (Miami, Fla.), and GenzymePharmaceuticals (Cambridge, Mass.). Atypical amino acids may bepurchased from commercial suppliers, synthesized de novo, or chemicallymodified or derivatized from naturally occurring amino acids.

As used herein an amino acid “substitution” refers to the replacement ofone amino acid residue by a different amino acid residue.

As used herein, the term “conservative amino acid substitution” isdefined herein as exchanges within one of the following five groups:

-   -   I. Small aliphatic, nonpolar or slightly polar residues:        -   Ala, Ser, Thr, Pro, Gly;    -   II. Polar, negatively charged residues and their amides:        -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;    -   III. Polar, positively charged residues:        -   His, Arg, Lys; Ornithine (Orn)    -   IV. Large, aliphatic, nonpolar residues:        -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine    -   V. Large, aromatic residues:        -   Phe, Tyr, Trp, acetyl phenylalanine

As used herein the general term “polyethylene glycol chain” or “PEGchain”, refers to mixtures of condensation polymers of ethylene oxideand water, in a branched or straight chain, represented by the generalformula H(OCH₂CH₂)_(n)OH, wherein n is at least 2. “Polyethylene glycolchain” or “PEG chain” is used in combination with a numeric suffix toindicate the approximate average molecular weight thereof. For example,PEG-5,000 refers to polyethylene glycol chain having a total molecularweight average of about 5,000 Daltons.

As used herein the term “pegylated” and like terms refers to a compoundthat has been modified from its native state by linking a polyethyleneglycol chain to the compound. A “pegylated polypeptide” is a polypeptidethat has a PEG chain covalently bound to the polypeptide.

As used herein a “linker” is a bond, molecule or group of molecules thatbinds two separate entities to one another. Linkers may provide foroptimal spacing of the two entities or may further supply a labilelinkage that allows the two entities to be separated from each other.Labile linkages include photocleavable groups, acid-labile moieties,base-labile moieties and enzyme-cleavable groups.

As used herein a “dimer” is a complex comprising two subunits covalentlybound to one another via a linker. The term dimer, when used absent anyqualifying language, encompasses both homodimers and heterodimers. Ahomodimer comprises two identical subunits, whereas a heterodimercomprises two subunits that differ, although the two subunits aresubstantially similar to one another.

The term “C₁-C_(n) alkyl” wherein n can be from 1 through 6, as usedherein, represents a branched or linear alkyl group having from one tothe specified number of carbon atoms. Typical C₁-C₆ alkyl groupsinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,butyl, iso-Butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The terms “C₂-C_(n) alkenyl” wherein n can be from 2 through 6, as usedherein, represents an olefinically unsaturated branched or linear grouphaving from 2 to the specified number of carbon atoms and at least onedouble bond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl (—CH₂—CH═CH₂), 1,3-butadienyl, (—CH═CHCH═CH₂),1-butenyl (—CH═CHCH₂CH₃), hexenyl, pentenyl, and the like.

The term “C₂-C_(n) alkynyl” wherein n can be from 2 to 6, refers to anunsaturated branched or linear group having from 2 to n carbon atoms andat least one triple bond. Examples of such groups include, but are notlimited to, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,and the like.

As used herein the term “aryl” refers to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and thelike. The size of the aryl ring and the presence of substituents orlinking groups are indicated by designating the number of carbonspresent. For example, the term “(C₁-C₃ alkyl)(C₆-C₁₀ aryl)” refers to a5 to 10 membered aryl that is attached to a parent moiety via a one tothree membered alkyl chain.

The term “heteroaryl” as used herein refers to a mono- or bi-cyclic ringsystem containing one or two aromatic rings and containing at least onenitrogen, oxygen, or sulfur atom in an aromatic ring. The size of theheteroaryl ring and the presence of substituents or linking groups areindicated by designating the number of carbons present. For example, theterm “(C₁-C_(n) alkyl)(C₅-C₆ heteroaryl)” refers to a 5 or 6 memberedheteroaryl that is attached to a parent moiety via a one to “n” memberedalkyl chain.

As used herein, the term “halo” refers to one or more members of thegroup consisting of fluorine, chlorine, bromine, and iodine.

As used herein the term “patient” without further designation isintended to encompass any warm blooded vertebrate domesticated animal(including for example, but not limited to livestock, horses, cats, dogsand other pets) and humans.

The term “isolated” as used herein means having been removed from itsnatural environment. In some embodiments, the analog is made throughrecombinant methods and the analog is isolated from the host cell.

The term “purified,” as used herein relates to the isolation of amolecule or compound in a form that is substantially free ofcontaminants normally associated with the molecule or compound in anative or natural environment and means having been increased in purityas a result of being separated from other components of the originalcomposition. The term “purified polypeptide” is used herein to describea polypeptide which has been separated from other compounds including,but not limited to nucleic acid molecules, lipids and carbohydrates.

A “peptidomimetic” refers to a chemical compound having a structure thatis different from the general structure of an existing peptide, but thatfunctions in a manner similar to the existing peptide, e.g., bymimicking the biological activity of that peptide. Peptidomimeticstypically comprise naturally-occurring amino acids and/or unnaturalamino acids, but can also comprise modifications to the peptidebackbone. For example a peptidomimetic may include a sequence ofnaturally-occurring amino acids with the insertion or substitution of anon-peptide moiety, e.g. a retroinverso fragment, or incorporation ofnon-peptide bonds such as an azapeptide bond (CO substituted by NH) orpseudo-peptide bond (e.g. NH substituted with CH2), or an ester bond(e.g., depsipeptides, wherein one or more of the amide (—CONHR—) bondsare replaced by ester (COOR) bonds). Alternatively the peptidomimeticmay be devoid of any naturally-occurring amino acids.

As used herein the term “FGF21-based analog” or “FGF21 analog” are usedinterchangeably, and absent further limitation define an FGF peptide ofSEQ ID NO: 173 modified to comprise a substitution at position 181 witha non-charged amino acid (i.e., excluding Asp, Glu, Lys, Arg and His)and optionally selected from the group consisting of Ala, Val, Gly, Thr,Cys, Pro, Met, Be and Leu, and optionally one or more of the followingmodifications:

i) one or more substitutions at positions selected from the groupconsisting of positions 157-161, 163, 166-168, 170-174 and 179-180(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173);or

ii) one or more substitutions selected from the group consisting ofA31C, G43C, L98D, L100K, N121D, and D127K (based on the numbering of themature FGF21 peptide of SEQ ID NO: 173); or

iii) a substitution of the native amino acid at position 167 and/or 175with the corresponding D-isomer of said native amino acid;

iv) substitution of the native C-terminal 25 amino acids of SEQ ID NO:173 with the sequence of SEQ ID NO: 188 or

v) and any combination of i) and ii) or i), ii) and iii) or acombination of ii) with iii).

As used herein the term “FGF21-based peptide conjugate” defines aconjugate comprising a modified peptide of SEQ ID NO: 166 linked to aconjugate moiety, wherein the modified peptide differs from the peptideof SEQ ID NO: 166 by one or more amino acid substitutions at positionsselected from positions 1, 2, 3, 4, 5, 7, 10, 11, 12, 14, 15, 16, 17,18, 23, 24 and 25 of SEQ ID NO: 166.

Abbreviations

Insulin analogs will be abbreviated as follows:

The insulin A and B chains will be designated generically by a capital Afor the A chain and a capital B for the B chain. When present, asuperscript 0 (e.g., A⁰ or B⁰) will designate the base sequence is aninsulin sequence (A chain: SEQ ID NO: 1, B chain SEQ ID NO: 2) and asuperscript 1 (e.g., A¹ or B¹) will designate the base sequence is anIGF-1 sequence (A chain: SEQ ID NO: 5, B chain SEQ ID NO: 6).Modifications that deviate from the native insulin and IGF sequence areindicated in parenthesis following the designation of the A or B chain(e.g., [B¹(H5,H10,Y16,L17): A¹(H8,N18,N21)]) with the single letteramino acid abbreviation indicating the substitution and the numberindicating the position of the substitution in the respective A or Bchain, using native insulin numbering. A colon between the A and B chainindicates a two chain insulin whereas a dash will indicate a covalentbond and thus a single chain analog. In single chain analogs a linkingmoiety will be included between the A and B chains and the designationC¹ refers to the native IGF 1 C peptide, SEQ ID NO: 17. The designation“position C8” in reference to the linking moiety designates an aminoacid located at the position corresponding to the eighth amino acid ofSEQ ID NO: 17.

Embodiments

The beneficial pharmacology observed in preclinical models indicate thatFGF21 and its analogs or mimetics hold promise as innovativetherapeutics for treating metabolic disorders. However, analogs of FGF21having greater potency at the FGF receptor are needed to enhance theefficacy of FGF21 mediated therapies.

At a molecular level, FGF21 interacts with the FGF receptor only intissues expressing the cofactor Klotho β. The Klotho β-dependent tissuespecificity is consistent with the predominant effects of FGF21occurring in liver and adipose tissue. Accordingly, one approach toenhance the potency of FGF21 analogs at the FGF21 receptor would be tomodify FGF21 to enhance its interaction with Klotho β.

The C-terminus of FGF21 is believed to play a key role in binding withKlotho β and applicants have demonstrated that a peptide comprising theC-terminal 25 amino acids of FGF21 (SEQ ID NO: 166) can inhibit activityof the native FGF21 peptide at its receptor. Presumably this antagonismarises from the affinity of the peptide for Klotho β. In one embodiment,the present disclosure is directed to peptides, and conjugatescomprising such peptides, that impact the activity of FGF21 at the FGF21receptor. More particularly, in one embodiment structural optimizationof the peptide of SEQ ID NO: 166 is performed to enhance the peptide'sinteraction with Klotho β.

In one embodiment a derivative of SEQ ID NO: 166 is provided wherein thederivative comprises a peptide that differs from SEQ ID NO: 166 by oneor more amino acid modifications, wherein said peptide has enhancedability to bind to Klotho β and/or enhanced ability to antagonize FGF21activity, relative to the peptide of SEQ ID NO: 166. In one embodiment apeptide is provided comprising the structure ofPPX₃X₄GX₆SX₈X₉X₁₀SX₁₂X₁₃GPSQGRX₂₀X₂₁SX₂₃AS (SEQ ID NO: 168) wherein X₃,X₄, X₆, X₈, X₉, X₁₀, X₁₂, X₁₃, X₂₀, X₂₁, and X₂₃ are independently anyamino acid, with the proviso that the peptide of SEQ ID NO: 168 differsfrom SEQ ID NO: 166 by at least one amino acid substitution. In oneembodiment one or more of X₃, X₄, X₆, X₈, X₉, X₁₀, X₁₂, X₁₃, X₂₀, X₂₁,and X₂₃ are amino acids in the D-stereo configuration, optionally theD-stereoisomer of the corresponding native amino acid at that position.

In accordance with one embodiment a peptide derivative of SEQ ID NO: 166is provided that differs from SEQ ID NO: 166 by 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 amino acid substitutions at any of amino acid positions 3, 4, 6,8, 9, 10, 12, 13, 20, 21 and 23 of SEQ ID NO: 166. In accordance withone embodiment a peptide derivative of SEQ ID NO: 166 is provided thatdiffers from SEQ ID NO: 166 by 1, 2, 3, 4, or 5 amino acid substitutionsat any of amino acid positions 3, 4, 6, 8, 9, 10, 12, 13, 20, 21 and 23of SEQ ID NO: 166. In accordance with one embodiment a peptidederivative of SEQ ID NO: 166 is provided that differs from SEQ ID NO:166 by 1 or 2 amino acid substitutions at any of amino acid positions 3,4, 6, 8, 9, 10, 12, 13, 20, 21 and 23 of SEQ ID NO: 166. In accordancewith one embodiment a peptide derivative of SEQ ID NO: 166 is providedthat differs from SEQ ID NO: 166 by 1, 2 or 3 amino acid substitutionsat amino acid positions selected from positions 8, 9 and 12 of SEQ IDNO: 166. In accordance with one embodiment a peptide derivative of SEQID NO: 166 is provided that differs from SEQ ID NO: 166 by 1 or 2 aminoacid substitutions at amino acid positions selected from positions 8, 9and 12 of SEQ ID NO: 166.

In one embodiment a peptide is provided comprising the structure ofPPDVGSSX₈X₉LSX₁₂VGPSQGRSPSYAS (SEQ ID NO: 169) wherein X₈, X₉, and X₁₂are independently any amino acid, with the proviso that the peptide ofSEQ ID NO: 169 differs from SEQ ID NO: 166 by at least one amino acidsubstitution. In one embodiment one or more of X₈, X₉, and X₁₂ are aminoacids in the D-stereo configuration, optionally the D-stereoisomer ofthe corresponding native amino acid at that position. In one embodimentX₈ is selected from the group consisting of Asp and D-Asp; X₉ isselected from the group consisting of Phe and D-Phe; and X₁₂ is selectedfrom the group consisting of Met and D-Met, with the proviso that thepeptide of SEQ ID NO: 169 differs from SEQ ID NO: 166 by at least oneamino acid substitution.

In one embodiment a peptide is provided comprising the structure ofPPDVGSSX₈X₉LSX₁₂VGPSQGRSPSYAS (SEQ ID NO: 169) wherein

X₈ is selected from the group consisting of D-Asp, α-methyl-L-asparticacid, α-methyl-D-aspartic acid;

X₉ is selected from the group consisting of D-Phe,D-4-t-Bu-phenylalanine (D-4-tBuPhe), D-alpha-methylphenylalanine(D-alpha-MePhe), D-4-biphenylalanine (D-4-Bip), D-1-naphthylalanine(D-1-Nal), D-2-naphthylalanine (D-2-Nal), 4-FPhe, 4-ClPhe, 4-BrPhe,4-IPhe, 4-NO₂Phe, and 3-NO₂Phe; and

X₁₂ is selected from the group consisting of D-Met, and methioninesulfoxide.

In accordance with one embodiment a peptide antagonist of Klotho βbinding/FGF21 activity is provided wherein the peptide is a derivativeof the C-terminal 25 amino acid sequence of FGF21. In particular,applicants have discovered that the C-terminal 25 amino acids of FGF21has equivalent antagonist activity for binding to Klotho β as a muchlarger fragment of FGF21 (amino acids 18-181). Furthermore, applicantshave discovered that by substituting the C-terminal amino acid of the25mer peptide fragment of FGF21, the potency of antagonism of thepeptide for FGF21 binding to Klotho β can be significantly enhanced. Inaccordance with one embodiment, a modified C-terminal 25 amino acidfragment of FGF21 (SEQ ID NO: 177) or a C-terminal 25 amino acidfragment of FGF19 (SEQ ID NO: 203) is provided wherein the C-terminalamino acid is substituted with a non-charged amino acid (e.g., excludingArg, Lys, Asp, Glu or His). This peptide can be further modified with 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid modifications. In oneembodiment the C-terminal amino acid of the FGF21 modified peptidefragment is an aliphatic amino acid selected from the group consistingof Gly, Ala, Val, Pro, Cys, Thr, Met, Leu and Be. In one embodiment theC-terminal amino acid of the FGF21 modified peptide fragment is analiphatic amino acid selected from the group consisting of Gly, Ala,Val, Pro, Met, Leu and Ile. In one embodiment the C-terminal amino acidof the FGF21 modified peptide fragment is an aliphatic amino acidselected from the group consisting of Gly, Ala, Val, Leu and Ile. In oneembodiment the C-terminal amino acid of the FGF21 modified peptidefragment is an aliphatic amino acid selected from the group consistingof Gly, Ala and Val. In one embodiment the C-terminal amino acid of theFGF21 modified peptide fragment is Ala.

In accordance with one embodiment peptides based on the C-terminal 25amino acids of native FGF21 and FGF19 are provided that have an enhancedability to bind Klotho β and can be used as antagonists of FGF receptoractivity. In one embodiment these peptides comprise a C-terminal 25amino acid fragment of FGF21 (SEQ ID NO: 177) that is modified by asubstitution at position 25 with a non-charged amino acid (e.g.,excluding Arg, Lys, Asp, Glu or His), optionally substituted withalanine, and optionally one or more modifications selected from:

i) one or more substitutions selected from the group consisting of G5L,G5F, G5S, S7M, L10F, S11G, M12L, P15R, Y23F, and A24E (based on thenumbering of the FGF21 peptide fragment of SEQ ID NO: 177); or

ii) one or more substitution of the native amino acid at position 11 or19 (based on the numbering of the FGF21 peptide fragment of SEQ ID NO:177) with the corresponding D-isomer of that amino acid; or

iii) and any combination of i) and ii).

In one embodiment a modified C-terminal 25 amino acid fragment of FGF21(SEQ ID NO: 177) is provided, wherein the modified peptide comprises asubstitution at position 25 with alanine, and

i) one or more substitutions selected from the group consisting of G5L,G5F, G5S, S7M, L10F, S11G, M12L, P15R, Y23F, and A24E.

In one embodiment a modified C-terminal 25 amino acid fragment of FGF21(SEQ ID NO: 177) is provided, wherein the modified peptide comprises asubstitution at position 25 with a non-charged amino acid (e.g.,excluding Arg, Lys, Asp, Glu or His), optionally substituted withalanine, and

i) one or more substitutions selected from the group consisting of G5L,G5F, G5S, S7M, L10F, S11G, M12L, P15R, Y23F, and A24E; and

ii) a substitution of the native amino acid at position 11 or 19 (basedon the numbering of the FGF21 peptide fragment of SEQ ID NO: 177) withthe corresponding D-isomer of that amino acid.

In one embodiment a modified C-terminal 25 amino acid fragment of FGF21(SEQ ID NO: 177) is provided, wherein the modified peptide comprises asubstitution at position 25 with a non-charged amino acid (e.g.,excluding Arg, Lys, Asp, Glu or His), optionally substituted withalanine, and one or more substitutions selected from the groupconsisting of S7M, L10F, S11G, and M12L.

In one embodiment a modified C-terminal 25 amino acid fragment of FGF21(SEQ ID NO: 177) is provided, wherein the modified peptide comprises asubstitution at position 25 with a non-charged amino acid (e.g.,excluding Arg, Lys, Asp, Glu or His), optionally substituted withalanine, and 2, 3, 4, 5, 6, 7 or 8 substitutions selected from the groupconsisting of G5L, G5F, G5S, S7M, L10F, S11G, M12L, P15R, Y23F, andA24E.

In one embodiment a modified C-terminal 25 amino acid fragment of FGF21(SEQ ID NO: 177) is provided, wherein the modified peptide comprises asubstitution at position 25 with a non-charged amino acid (e.g.,excluding Arg, Lys, Asp, Glu or His), optionally substituted withalanine, and further substitutions of S7M, L10F, S11G, M12L, P15R, Y23F,and A24E.

In accordance with one embodiment peptides based on the C-terminal 25amino acids of native FGF21 and FGF19 are provided that have an enhancedability to bind Klotho β and can function as antagonists of FGF receptoractivity. In accordance with one embodiment a 25 amino acid peptideantagonist of FGF21 receptor activity is selected from the groupconsisting of SEQ ID NOs: 179, 180, 188, 191, 234, 237, 238, 239, 240 or241. Each of these peptides have been found to be more potent inantagonizing the native FGF21's in vitro signaling than thecorresponding native FGF21 C-terminal 25 amino acid fragment.

As demonstrated by the data presented in Examples 1 and 2 certainpositions of the 25 amino acid C-terminal peptide are tolerant of aminoacid substitutions without substantial impact to the ability of thepeptide to bind Klotho β and/or inhibit FGF21 activity at the FGF21receptor. In particular positions 1, 2, 5, 7, 11, 14, 15, 16, 17, 18 and19 of the C-terminal FGF21 fragment tolerate amino acid substitutionswithout substantial loss in their ability to antagonizing the nativeFGF21's in vitro signaling. In accordance with one embodiment aderivative of a peptide comprising SEQ ID NO: 188, SEQ ID NO: 191, SEQID NO: 180, SEQ ID NO: 179 or SEQ ID NO: 234 is provided, wherein thederivative peptide differs from SEQ ID NO: 188, SEQ ID NO: 191, SEQ IDNO: 180, SEQ ID NO: 179 or SEQ ID NO: 234 by 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 amino acid substitutions selected from positions 1, 2, 5, 7,11, 14, 15, 16, 17, 18 and 19 relative to those sequences. In oneembodiment the derivative peptide differs by 1, 2 or 3 amino acidsubstitutions selected from positions 1, 2, 5, 7, 11, 14, 15, 16, 17, 18and 19. In one embodiment the amino acid substitutions at positions 1,2, 5, 7, 11, 14, 15, 16, 17, 18 and 19 are conservative amino acidsubstitutions. In one embodiment a derivative of a peptide comprisingSEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 180, SEQ ID NO: 179 or SEQ IDNO: 234 is provided, wherein the derivative peptide differs from SEQ IDNO: 188, SEQ ID NO: 191, SEQ ID NO: 180, SEQ ID NO: 179 or SEQ ID NO:234 by 1, 2 or 3 amino acid substitutions selected from positions 1, 2,5, 14, 15, 16, 17, 18 and 19, optionally wherein the amino acidsubstitutions are conservative amino acid substitutions. In oneembodiment a derivative of a peptide comprising SEQ ID NO: 188, SEQ IDNO: 191, SEQ ID NO: 180, SEQ ID NO: 179 or SEQ ID NO: 234 is provided,wherein the derivative peptide differs from SEQ ID NO: 188, SEQ ID NO:191, SEQ ID NO: 180, SEQ ID NO: 179 or SEQ ID NO: 234 by 1, 2 or 3 aminoacid substitutions selected from positions 2, 5, 14, 15, 16, 17, 18 and19, optionally wherein the amino acid substitutions are conservativeamino acid substitutions. In one embodiment a peptide with antagonistactivity against Klotho β is provided wherein the peptide comprises anamino acid sequence ofX₁X₂X₃X₄X₅SX₇DPX₁₀X₁₁X₁₂VX₁₄GX₁₆X₁₇X₁₈X₁₉RSPSX₂₄X₂₅X₂₆ (SEQ ID NO: 235),

wherein

X₁ is Pro or absent;

X₂ is Pro or Leu;

X₃ is Asp or Glu;

X₄ is Val or Thr;

X₅ is Gly, Asp, Phe, Leu or Ser;

X₇ is Ser or Met;

X₁₀ is Leu or Phe;

X₁₁ is Ser or Gly;

X₁₂ is Met or Leu;

X₁₄ is absent or Thr;

X₁₆ is Pro, Leu, Arg, Glu, or Gly;

X₁₇ is Ser or Glu;

X₁₈ is Gln or Ala;

X₁₉ is Gly or Val;

X₂₄ is Tyr or Phe;

X₂₅ is Ala or Glu; and

X₂₆ is an aliphatic amino acid selected from Gly, Ala, Val, Leu, Ser, orIle, optionally comprising up to 5 (i.e., 1, 2, 3, 4 or 5) further aminoacid substitutions. Optionally the up to 5 further modification caninclude additional amino acid substitutions at any of positions 1-5, 7,10-12, 14, 16-19 or 24-26 of SEQ ID NO: 235, or at positions 1, 2, 3, 5,7, 11, 14, 15, 16, 17, 18 and 19 of SEQ ID NO: 235, or optionally thepeptide comprises up to 2 further amino acid substitutions at positionsselected from positions 15 and 23 of SEQ ID NO: 235. In one embodimentX₁ is absent and X₁₄ is Thr. In one embodiment X₁₆ is Pro, Leu, or Arg.In one embodiment a peptide with antagonist activity against Klotho β isprovided wherein the peptide comprises an amino acid sequence ofX₁X₂X₃X₄X₅SX₇DPX₁₀X₁₁X₁₂VX₁₄GX₁₆X₁₇X₁₈X₁₉RSPSX₂₄X₂₅A (SEQ ID NO: 236),

wherein

X₁ is Pro or absent;

X₂ is Pro or Leu;

X₃ is Asp or Glu;

X₄ is Val or Thr;

X₅ is Gly, Asp, Phe, Leu or Ser;

X₇ is Ser or Met;

X₁₀ is Leu or Phe;

X₁₁ is Ser or Gly;

X₁₂ is Met or Leu;

X₁₄ is absent or Thr;

X₁₆ is Pro, Leu, or Arg;

X₁₇ is Ser or Glu;

X₁₈ is Gln or Ala;

X₁₉ is Gly or Val;

X₂₄ is Tyr or Phe;

X₂₅ is Ala or Glu; and

In accordance with one embodiment a pharmaceutical composition isprovided comprising an FGF peptide fragment antagonist as disclosedherein and a pharmaceutically acceptable carrier, diluent, or excipient.

Each of the 25 amino acid FGF21 C-terminal peptide fragments, analogsand derivatives disclosed herein can be linked to the carboxy terminusof a larger peptide to form a contiguous polypeptide sequence. Forexample each of the 25 amino acid FGF21 peptide fragments, analogs andderivatives disclosed herein can be fused to the carboxy terminus of aN-terminal polypeptide fragment of FGF21 (or analog thereof) includingfor example a peptide selected from the group consisting of SEQ ID NO:194, SEQ ID NO: 195 and SEQ ID NO: 196, or a peptide that differs fromSEQ ID NO: 194, SEQ ID NO: 195 and SEQ ID NO: 196 by 1, 2, 3, 4, 5, 6,7, 8, or 9 amino acid substitutions, to form a polypeptide havingagonist activity at the FGF21 receptor. In one embodiment the 1, 2, 3,4, 5, 6, 7, 8, or 9 amino acid substitutions are conservative amino acidsubstitutions.

Substituting the novel sequences of SEQ ID NOs: 179, 180, 234, 188 or191 for the native C-terminal 25 amino acids of FGF21 or FGF19 producesan FGF analog that has higher potency at the FGF receptor than nativeFGF2. Accordingly, one aspect of the present disclosure is directed toan FGF21 analog comprising the sequence of

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVG

GAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEA

CSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQLETDSMDPFGLVTGLEAVRSPSFEA (SEQ ID NO: 192); or

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVG

GAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEA

CSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAA (SEQ ID NO: 206); or

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVG

GAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEA

CSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 207); or

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVG

GAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEA

CSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPLETDSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 208); or

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVG

GAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEA

CSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPDVGSMDPFGLVTGLEAVRSPSYAA (SEQ ID NO: 209). Each of thepeptides of SEQ ID NOs: 192, 206, 207, 208 and 209 can be furthermodified to enhance the potency of the FGF21 analog as an FGF receptoragonist. In one embodiment the peptides of SEQ ID NOs: 192, 206, 207,208 and 209 are further modified to comprise

i) one or more substitutions selected from the group consisting of A31C,G43C, L98D, L100K, N121D, and D127K (based on the numbering of themature FGF21 sequence of SEQ ID NO: 173); or

ii) a substitution of the native amino acid at position 167 and/or 175with the corresponding D-isomer of that amino acid (based on thenumbering of the mature FGF21 sequence of SEQ ID NO: 173); or

iii) any combination of i) or ii).

In accordance with one embodiment an FGF21 analog having enhancedactivity at the FGF receptor relative to native FGF (based on the Erk1/2phosphorylation in 293T HEK hKLB cell assay of Example 1) is provided.In one embodiment the FGF21 analog comprises the sequence of SEQ ID NO:175 or SEQ ID NO: 176.

In accordance with one embodiment analogs of FGF21 are provided whereinthe analog has higher potency at the FGF receptor relative to native FGF(based on the Erk1/2 phosphorylation in 293T HEK hKLB cell assay ofExample 1). In accordance with one embodiment an FGF21-based analog isprovided that differs from the FGF peptide of SEQ ID NO: 173 by asubstitution at position 181 with a non-charged amino acid (i.e.,excluding Asp, Glu, Lys, Arg and His), and optionally an amino acidselected from the group consisting of Ala, Val, Gly, Thr, Cys, Pro, Met,Ile and Leu, and optionally one or more of the following modifications:

i) one or more substitutions selected from the group consisting ofG161L, G161F, G161S, S163M, L166F, S167G, M168L, P171R, Y179F, and A180E(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173);or

ii) one or more substitutions selected from the group consisting ofA31C, G43C, L98D, L100K, N121D, and D127K (based on the numbering of themature FGF21 peptide of SEQ ID NO: 173); or

iii) a substitution of the native amino acid at position 167 and/or 175with the corresponding D-isomer of that amino acid (based on thenumbering of the mature FGF21 peptide of SEQ ID NO: 173);

iv) substitution of the native C-terminal 25 amino acids of SEQ ID NO:173 with the sequence of SEQ ID NO: 188 or

v) any combination of i) and ii), any combination of i), ii) and iii) orany combination of ii) with iv). Optionally the amino acid at position181 is selected from the group consisting of Ala, Val, Gly, Ile and Leu,or is selected from the group consisting of Ala, Val, Ile and Leu, or isselected from the group consisting of Ala and Val.

In one embodiment the FGF21-based analog peptides comprise a modifiedpeptide of SEQ ID NO: 204, wherein the modified peptide comprises one ormore modifications selected from:

i) one or more substitutions selected from the group consisting ofG161L, G161F, G161S, S163M, L166F, S167G, M168L, P171R, Y179F, and A180E(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173);or

ii) one or more substitutions selected from the group consisting ofA31C, G43C, L98D, L100K, N121D, and D127K (based on the numbering of themature FGF21 peptide of SEQ ID NO: 173); or

iii) a substitution of the native amino acid at position 167 and/or 175(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173)with the corresponding D-isomer of that amino acid; or

iv) any combination of i) ii), and iii).

In one embodiment a modified analog of FGF21 comprising the sequence ofSEQ ID NO: 204 is provided, wherein the modified peptide differs fromSEQ ID NO: 204 by comprising

i) one or more substitutions selected from the group consisting ofG161L, G161F, G161S, S163M, L166F, S167G, M168L, P171R, Y179F, and A180E(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173);and

ii) one or more substitutions selected from the group consisting ofA31C, G43C, L98D, L100K, N121D, and D127K (based on the numbering of themature FGF21 peptide of SEQ ID NO: 173).

In one embodiment a modified analog of FGF21 comprising the sequence ofSEQ ID NO: 204 is provided, wherein the modified peptide differs fromSEQ ID NO: 204 by comprising

i) one or more substitutions selected from the group consisting ofG161L, G161F, G161S, S163M, L166F, S167G, M168L, P171R, Y179F, and A180E(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173);and

ii) one or more substitutions selected from the group consisting ofA31C, G43C, L98D, L100K, N121D, and D127K (based on the numbering of themature FGF21 peptide of SEQ ID NO: 173); and

iii) a substitution of the native amino acid at position 167 and/or 175(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173)with the corresponding D-isomer of that amino acid.

In one embodiment a modified analog of FGF21 comprising the sequence ofSEQ ID NO: 204 is provided, wherein the modified peptide differs fromSEQ ID NO: 204 by comprising

i) one or more substitutions selected from the group consisting ofS163M, L166F, S167G, M168L (based on the numbering of the mature FGF21peptide of SEQ ID NO: 173); and

ii) a substitution of the native amino acid at position 167 and/or 175(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173)with the corresponding D-isomer of that amino acid.

In one embodiment a modified analog of FGF21 comprising the sequence ofSEQ ID NO: 204 is provided, wherein the modified peptide differs fromSEQ ID NO: 204 by comprising

i) one or more substitutions selected from the group consisting ofG161L, G161F, G161S, S163M, L166F, S167G, M168L, P171R, Y179F, and A180E(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173).

In one embodiment a modified analog of FGF21 comprising the sequence ofSEQ ID NO: 204 is provided, wherein the modified peptide differs fromSEQ ID NO: 204 by comprising

i) 2, 3, 4, 5, 6 or 7 substitutions selected from the group consistingof G161L, G161F, G161S, S163M, L166F, S167G, M168L, P171R, Y179F, andA180E (based on the numbering of the mature FGF21 peptide of SEQ ID NO:173); and

ii) one or more substitutions selected from the group consisting ofA31C, G43C, L98D, L100K, N121D, and D127K (based on the numbering of themature FGF21 peptide of SEQ ID NO: 173).

In one embodiment a modified analog of FGF21 comprising the sequence ofSEQ ID NO: 204 is provided, wherein the modified peptide differs fromSEQ ID NO: 204 by comprising

i) substitutions of S163M, L166F, S167G, M168L, P171R, Y179F, and A180E(based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173);and

ii) one or more substitutions selected from the group consisting ofA31C, G43C, L98D, L100K, N121D, and D127K (based on the numbering of themature FGF21 peptide of SEQ ID NO: 173).

In one embodiment a modified analog of FGF21 comprising the sequence ofSEQ ID NO: 204 is provided, wherein the modified peptide differs fromSEQ ID NO: 204 by comprising

i) substitutions of S163M, L166F, S167G, M168L, P171R, Y179F, and A180Eand one of G161L, G161F and G161S (based on the numbering of the matureFGF21 peptide of SEQ ID NO: 173); and

ii) one or more substitutions selected from the group consisting ofA31C, G43C, L98D, L100K, N121D, and D127K (based on the numbering of themature FGF21 peptide of SEQ ID NO: 173).

In one embodiment a modified analog of FGF21 comprising the sequence ofSEQ ID NO: 204 is provided, wherein the modified peptide differs fromSEQ ID NO: 204 by comprising

i) substitutions of S163M, L166F, S167G, M168L, P171R, Y179F, and A180Eand one of G161L, G161F and G161S (based on the numbering of the matureFGF21 peptide of SEQ ID NO: 173); and

ii) substitutions of A31C, G43C, L98D, L100K, N121D, and D127K (based onthe numbering of the mature FGF21 peptide of SEQ ID NO: 173). Inaccordance with one embodiment an FGF21-based analog is provided whereinthe analog comprises a sequence selected from the group consisting ofSEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 204, SEQ ID NO: 205, SEQ IDNO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251and SEQ ID NO: 252. In accordance with one embodiment an analog of FGF21is provided wherein the analog comprises a peptide sequence selectedfrom the group consisting of SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO:249, SEQ ID NO: 250, SEQ ID NO: 251 and SEQ ID NO: 252. In accordancewith one embodiment an analog of FGF21 is provided wherein the analogcomprises the sequence of SEQ ID NO: 193 or SEQ ID NO: 205. Inaccordance with one embodiment an analog of FGF21 is provided whereinthe analog consists of the sequence of SEQ ID NO: 193. In accordancewith one embodiment an analog of FGF21 is provided wherein the analogconsists of the sequence of SEQ ID NO: 205. In accordance with oneembodiment an analog of FGF21 is provided wherein the analog consists ofthe sequence of SEQ ID NO: 248.

The FGF21 analogs disclosed herein as having high potency at the FGFreceptor can be used for any previous use identified for native FGF21.This includes but is not limited to the treatment of a disease ormedical condition selected from the group consisting of metabolicsyndrome, diabetes, obesity, liver steatosis, dyslipidemia, and chroniccardiovascular disease.

In accordance with one embodiment a pharmaceutical composition isprovided comprising an FGF21 agonist analog as disclosed herein and apharmaceutically acceptable carrier, diluent, or excipient. Inaccordance with one embodiment the pharmaceutical composition can beused for reducing weight gain or inducing weight loss in a patient inneed thereof or for reducing elevated triglycerides, improvinghyperglycemia and treating diabetes.

In some aspects, the invention provides a pharmaceutical compositioncomprising any of the novel FGF21 analogs disclosed herein, preferablysterile and preferably at a purity level of at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptablediluent, carrier or excipient. Such compositions may contain an FGF21analog peptide at a concentration of at least A, wherein A is 0.001mg/ml, 0.01 mg/ml, 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml,12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml orhigher. In other embodiments, such compositions may contain an FGF21analog at a concentration of at most B, wherein B is 30 mg/ml, 25 mg/ml,24 mg/ml, 23, mg/ml, 22 mg/ml, 21 mg/ml, 20 mg/ml, 19 mg/ml, 18 mg/ml,17 mg/ml, 16 mg/ml, 15 mg/ml, 14 mg/ml, 13 mg/ml, 12 mg/ml, 11 mg/ml 10mg/ml, 9 mg/ml, 8 mg/ml, 7 mg/ml, 6 mg/ml, 5 mg/ml, 4 mg/ml, 3 mg/ml, 2mg/ml, 1 mg/ml, or 0.1 mg/ml. In some embodiments, the compositions maycontain an FGF21 analog at a concentration range of A to B mg/ml, forexample, 0.001 to 30.0 mg/ml. In one embodiment the pharmaceuticalcompositions comprise aqueous solutions that are sterilized andoptionally stored within various containers. The compounds of thepresent invention can be used in some embodiments to preparepre-formulated solutions ready for injection. In other embodiments thepharmaceutical compositions comprise a lyophilized powder. Thepharmaceutical compositions can be further packaged as part of a kitthat includes a disposable device for administering the composition to apatient. The containers or kits may be labeled for storage at ambientroom temperature or at refrigerated temperature.

The FGF21 analog peptides can be administered to a patient using anystandard route of administration, including parenterally, such asintravenously, intraperitoneally, subcutaneously or intramuscularly,intrathecally, transdermally, rectally, orally, nasally or byinhalation. In one embodiment the composition is administeredsubcutaneously or intramuscularly.

In one embodiment the kit is provided with a device for administeringthe FGF21 analog composition to a patient, e.g. syringe needle, pendevice, jet injector or other needle-free injector. The kit mayalternatively or in addition include one or more containers, e.g.,vials, tubes, bottles, single or multi-chambered pre-filled syringes,cartridges, infusion pumps (external or implantable), jet injectors,pre-filled pen devices and the like, optionally containing the FGF21analog in a lyophilized form or in an aqueous solution. Preferably, thekits will also include instructions for use. In some embodiments thedevice of the kit is an aerosol dispensing device, wherein thecomposition is prepackaged within the aerosol device. In anotherembodiment the kit comprises a syringe and a needle, and in oneembodiment the sterile composition comprising the FGF21 analog isprepackaged within the syringe.

In accordance with one embodiment a pharmaceutical composition isprovided wherein the composition comprises an FGF21 analog of thepresent disclosure, or pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. The pharmaceutical composition cancomprise any pharmaceutically acceptable ingredient, including, forexample, acidifying agents, additives, adsorbents, aerosol propellants,air displacement agents, alkalizing agents, anticaking agents,anticoagulants, antimicrobial preservatives, antioxidants, antiseptics,bases, binders, buffering agents, chelating agents, coating agents,coloring agents, desiccants, detergents, diluents, disinfectants,disintegrants, dispersing agents, dissolution enhancing agents, dyes,emollients, emulsifying agents, emulsion stabilizers, fillers, filmforming agents, flavor enhancers, flavoring agents, flow enhancers,gelling agents, granulating agents, humectants, lubricants,mucoadhesives, ointment bases, ointments, oleaginous vehicles, organicbases, pastille bases, pigments, plasticizers, polishing agents,preservatives, sequestering agents, skin penetrants, solubilizingagents, solvents, stabilizing agents, suppository bases, surface activeagents, surfactants, suspending agents, sweetening agents, therapeuticagents, thickening agents, tonicity agents, toxicity agents,viscosity-increasing agents, water-absorbing agents, water-misciblecosolvents, water softeners, or wetting agents. The instant compositionsmay further comprise, for example, micelles or liposomes, or some otherencapsulated form, or may be administered in an extended release form toprovide a prolonged storage and/or delivery effect. In certainembodiments, the pharmaceutical compositions may comprise bufferingagents to achieve a physiological compatible pH.

The disclosed pharmaceutical formulations may be administered accordingto any regime including, for example, daily (1 time per day, 2 times perday, 3 times per day, 4 times per day, 5 times per day, 6 times perday), every two days, every three days, every four days, every fivedays, every six days, weekly, bi-weekly, every three weeks, monthly, orbi-monthly.

In some embodiments, a method of treating hyperglycemia, or a method ofreducing weight gain or inducing weight loss in a patient is provided,which involves administering to the patient an effective amount of anaqueous solution comprising an FGF21 analog as disclosed herein. Infurther embodiments, methods of treating diabetes involvingco-administering a conventional dose or a reduced dose of insulin and anFGF21 analog as disclosed herein are provided. Methods of treatingdiabetes with an FGF21 analog of the present disclosure, withoutco-administering insulin are also provided.

Methods for treating hyperglycemia are expected to be useful for avariety of types of hyperglycemia, including diabetes, diabetes mellitustype I, diabetes mellitus type II, or gestational diabetes, eitherinsulin-dependent or non-insulin-dependent, and reducing complicationsof diabetes including nephropathy, retinopathy and vascular disease.

Methods for reducing appetite or promoting loss of body weight areexpected to be useful in reducing body weight, preventing weight gain,or treating obesity of various causes, including drug-induced obesity,and reducing complications associated with obesity including vasculardisease (coronary artery disease, stroke, peripheral vascular disease,ischemia reperfusion, etc.), hypertension, onset of diabetes type II,hyperlipidemia and musculoskeletal diseases.

Metabolic Syndrome, also known as metabolic syndrome X, insulinresistance syndrome or Reaven's syndrome, is a disorder that affectsover 50 million Americans. Metabolic Syndrome is typically characterizedby a clustering of at least three or more of the following risk factors:(1) abdominal obesity (excessive fat tissue in and around the abdomen),(2) atherogenic dyslipidemia (blood fat disorders including hightriglycerides, low HDL cholesterol and high LDL cholesterol that enhancethe accumulation of plaque in the artery walls), (3) elevated bloodpressure, (4) insulin resistance or glucose intolerance, (5)prothrombotic state (e.g. high fibrinogen or plasminogen activatorinhibitor-1 in blood), and (6) pro-inflammatory state (e.g. elevatedC-reactive protein in blood). Other risk factors may include aging,hormonal imbalance and genetic predisposition.

In accordance with one embodiment, a method of preventing or treatingMetabolic Syndrome, or reducing one, two, three or more risk factorsthereof, in a subject, is provided wherein the method comprisesadministering to the subject an FGF21 analog described herein in anamount effective to prevent or treat Metabolic Syndrome, or one or morerisk factors thereof.

Nonalcoholic fatty liver disease (NAFLD) refers to a wide spectrum ofliver disease ranging from simple fatty liver (steatosis), tononalcoholic steatohepatitis (NASH), to cirrhosis (irreversible,advanced scarring of the liver). All of the stages of NAFLD have incommon the accumulation of fat (fatty infiltration) in the liver cells(hepatocytes). Simple fatty liver is the abnormal accumulation of acertain type of fat, triglyceride, in the liver cells with noinflammation or scarring. In NASH, the fat accumulation is associatedwith varying degrees of inflammation (hepatitis) and scarring (fibrosis)of the liver. The inflammatory cells can destroy the liver cells(hepatocellular necrosis). In the terms “steatohepatitis” and“steatonecrosis”, steato refers to fatty infiltration, hepatitis refersto inflammation in the liver, and necrosis refers to destroyed livercells. Accordingly, the present disclosure also provides a method ofpreventing or treating Alcoholic Liver Disease, NAFLD, or any stagethereof, in a subject comprising administering to a subject an FGF21analog described herein in an amount effective to prevent or treatAlcoholic Liver Disease, NAFLD, or any stage thereof. Such treatmentmethods include reduction in one, two, three or more of the following:liver fat content, incidence or progression of cirrhosis, incidence ofhepatocellular carcinoma, signs of inflammation, e.g. abnormal hepaticenzyme levels (e.g., aspartate aminotransferase AST and/or alanineaminotransferase ALT, or LDH), elevated serum ferritin, elevated serumbilirubin, and/or signs of fibrosis, e.g. elevated TGF-beta levels.

The FGF21 analogs disclosed herein may be administered alone or incombination with other anti-diabetic or anti-obesity agents.Anti-diabetic agents known in the art or under investigation includeinsulin, sulfonylureas, such as tolbutamide (Orinase), acetohexamide(Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide(Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride(Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide(Prandin) or nateglinide (Starlix); biguanides such as metformin(Glucophage) or phenformin; thiazolidinediones such as rosiglitazone(Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or otherPPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydratedigestion, such as miglitol (Glyset), acarbose (Precose/Glucobay);exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP-4)inhibitors such as vildagliptin or sitagliptin; SGLT (sodium-dependentglucose transporter 1) inhibitors; glucokinase activators (GKA);glucagon receptor antagonists (GRA); or FBPase (fructose1,6-bisphosphatase) inhibitors.

Anti-obesity agents known in the art or under investigation include,Leptin and Fibroblast Growth Factor 21 (FGF-21), appetite suppressants,such as phenethylamine type stimulants, phentermine (optionally withfenfluramine or dexfenfluramine), diethylpropion (Tenuate®),phendimetrazine (Prelu-2®, Bontril®), benzphetamine (Didrex®),sibutramine (Meridia®, Reductil®); rimonabant (Acomplia®), othercannabinoid receptor antagonists; oxyntomodulin; fluoxetinehydrochloride (Prozac); Qnexa (topiramate and phentermine), Excalia(bupropion and zonisamide) or Contrave (bupropion and naltrexone); orlipase inhibitors, similar to xenical (Orlistat) or Cetilistat (alsoknown as ATL-962), or GT 389-255.

Additional conjugates can be formed between the FGF21 analogs disclosedherein (and peptide fragments thereof) and other bioactive peptides suchas insulin or nuclear hormones to mediated selective delivery of theconjugates to the liver.

In accordance with one embodiment a peptide conjugate is providedcomprising the C-terminal 25 amino acids of FGF21 (SEQ ID NO: 166) or aderivative of that sequence linked to a bioactive agent. In oneembodiment the conjugate comprises a compound of the general formula:Q-L-Y, wherein Q is a bioactive peptide, including for example a peptideselected from the group consisting of insulin, FGF1, FGF2, and a nuclearhormone; Y is a peptide comprising the sequence of SEQ ID NO: 166 or amodified peptide that differs from SEQ ID NO: 166 by 1, 2, 3, 4 or 5amino acid substitution at amino acid positions selected from any ofpositions 3, 4, 6, 8, 9, 10, 12, 13, 20, 21 and 23 of SEQ ID NO: 166;and L is a linking group or a bond. In accordance with one embodiment Yis a peptide comprising a sequence selected from the group consisting of

LETDSMDPFGLVTGLEAVRSPSFEA (SEQ ID NO: 188),

PPDVGSSDPLSMVGPSQGRSPSYAA (SEQ ID NO: 191),

PPDVGSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 180),

PLETDSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 179),

PDVGSMDPFGLVTGLEAVRSPSYAA (SEQ ID NO: 234),

PPDVG SMDPF GLVGR SQGRS PSFEA (SEQ ID NO: 237),

PPDVF SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 238),

PPDVL SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 239),

PPDVS SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 240), and

PPDVG SSDPF GLVGP SQGRS PSFEA (SEQ ID NO: 241).

In accordance with one embodiment an FGF21 analog is provided comprisingthe sequence of SEQ ID NO: 167 modified by one or more amino acidsubstitutions at positions selected from the group consisting ofpositions 159, 160, 162, 164, 165,166, 168, 169, 176, 177, and 179relative to SEQ ID NO: 167. In one embodiment the substitutions arereplacement of the native amino acid with its correspondingD-stereoisomer. In an alternative embodiment the substitutions arereplacement of the native amino acid with a corresponding amino acidmimetic of the native amino acid at that position.

In one embodiment conjugates of the FGF21 C-terminal peptide (SEQ ID NO:166 and derivative disclosed herein) and other bioactive peptides (e.g.,FGF1, FGF2 and insulin) are prepared as a means of directing theactivity of those bioactive peptides to adipose and liver. Conjugates ofthe FGF21 based peptide can also be used to restrict the pharmacology ofnuclear hormones as a means to enhance their safety without lesseningtheir proven pharmacology, thus rendering them suitable for chronic use.

Disclosed herein are FGF21 based peptides conjugated comprising aninsulin-like peptide, insulin peptide or a NHR ligand. In one embodimentthe NHR ligand is an NHR agonist. In one embodiment the NHR ligand isselected from the group consisting of a steroid that exhibits an EC₅₀ ofabout 1 μM or less when unconjugated to Q-L, and has a molecular weightof up to about 1000 daltons. In one embodiment the NHR ligand is aligand that activates the thyroid hormone receptor or a ligand thatactivates the peroxisome proliferator-activated receptors (PPAR).

In one embodiment Q is a glucagon peptide comprising a sequence from thegroup consisting of

HX₁QGTFTSDKSKYLDX₂RAAQDFVQWLMDT (SEQ ID NO: 202,

X₃AQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS (SEQ ID NO: 197),

X₄AQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS (SEQ ID NO: 198),

X₅AQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS (SEQ ID NO: 199),

X₆AQGTFTSDKSKYLDERAAQDFVQWLLDAGPSSGAPPPS (SEQ ID NO: 200) and

X₇AQGTFTSDKSKYLDERAAQDFVQWLLEAGPSSGAPPPS (SEQ ID NO: 201)

wherein

X₁ and X₂ are both Aib;

X₃ is Acetyl D-Tyr;

X₄ is Acetyl D-His;

X₅ is Acetyl D-thio Ala, and

X₆ and X₇ are both acetyl-D-Tyr

In some embodiments the NHR ligand is an NHR agonist. In one embodimentthe NHR agonist has activity at a Type I NHR when bound to Q-L. In oneembodiment the NHR agonist has activity at a Type II NHR when bound toQ-L. In one embodiment the NHR ligand is

-   -   i) a steroid that exhibits an EC₅₀ of about 1 μM or less when        unconjugated to Q-L, and further has a molecular weight of up to        about 1000 daltons; or    -   ii) a ligand that activates the thyroid hormone receptor; or    -   iii) a ligand that activates the peroxisome        proliferator-activated receptors (PPAR).

In one embodiment the NHR ligand of the conjugate is selected from thegroup consisting of estradiol and derivatives thereof, estrone andderivatives thereof, testosterone and derivatives thereof, and cortisoland derivatives thereof. In one embodiment the NHR ligand isdexamethasone.

In accordance with one embodiment the NHR ligand of the FGF21 basedconjugate is a thyroid hormone receptor agonist having the generalstructure

wherein

R₁₅ is C₁-C₄ alkyl, —CH₂(pyridazinone), —CH₂(OH)(phenyl)F, —CH(OH)CH₃,halo or H;

R₂₀ is halo, CH₃ or H;

R₂₁ is halo, CH₃ or H;

R₂₂ is H, OH, halo, —CH₂(OH)(C₆ aryl)F, or C₁-C₄ alkyl; and

R₂₃ is —CH₂CH(NH₂)COOH, —OCH₂COOH, —NHC(O)COOH, —CH₂COOH,

—NHC(O)CH₂COOH, —CH₂CH₂COOH, or —OCH₂PO₃ ²⁻. In one embodiment thethyroid hormone receptor agonist has the general structure of Formula I:

wherein

R₂₀, R₂₁, and R₂₂ are independently selected from the group consistingof H, OH, halo and C₁-C₄ alkyl; and

R₁₅ is halo or H. In one embodiment the thyroid hormone receptor agonistis selected from the group consisting of thyroxine T4(3,5,3′,5′-tetra-iodothyronine), and 3,5,3′-triiodo L-thyronine.

In one embodiment the NHR ligand is an agonist of a PPAR. In oneembodiment the PPAR agonist is selected from the group consisting ofTesaglitazar, Aleglitazar and thiazolidinediones. In one embodiment thePPAR agonist is Tesaglitazar or Aleglitazar.

In one embodiment the insulin-like peptide of the FGF21 based conjugateQ-L-Y is a peptide selected from IGF I, IGF II, an insulin like peptide3, 4, 5 or 6, or a Relaxin-1, 2 or 3. In one embodiment the insulin-likepeptide of the FGF21 based conjugate Q-L-Y is an insulin like peptide ora Relaxin peptide.

In one embodiment the insulin peptide of the FGF21 based conjugate Q-L-Yis a native insulin peptide or any insulin receptor agonist known tothose skilled in the art. In one embodiment the insulin peptide (Q)comprises an A chain and a B chain wherein said A chain comprises asequence

GIVX₄X₅CCX₈X₉X₁₀CX₁₂LX₁₄X₁₅LX₁₇X₁₈YCX₂₁—R₅₃ (SEQ ID NO: 19), and said Bchain comprises a sequence R₆₂-X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGX₄₁X₄₂GFX₄₅(SEQ ID NO: 20), wherein

-   -   X₄ is glutamic acid or aspartic acid;    -   X₅ is glutamine or glutamic acid    -   X₈ is histidine, threonine or phenylalanine;    -   X₉ is serine, arginine, lysine, ornithine or alanine;    -   X₁₀ is isoleucine or serine;    -   X₁₂ is serine or aspartic acid;    -   X₁₄ is tyrosine, arginine, lysine, ornithine or alanine;    -   X₁₅ is glutamine, glutamic acid, arginine, alanine, lysine,        ornithine or leucine;    -   X₁₇ is glutamic acid, aspartic acid, asparagine, lysine,        ornithine or glutamine;    -   X₁₈ is methionine, asparagine, glutamine, aspartic acid,        glutamic acid or threonine;    -   X₂₁ is selected from the group consisting of alanine, glycine,        serine, valine, threonine, isoleucine, leucine, glutamine,        glutamic acid, asparagine, aspartic acid, histidine, tryptophan,        tyrosine, and methionine;    -   X₂₅ is histidine or threonine;    -   X₂₉ is selected from the group consisting of alanine, glycine        and serine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid;    -   X₃₃ is selected from the group consisting of aspartic acid and        glutamic acid;    -   X₃₄ is selected from the group consisting of alanine and        threonine;    -   X₄₁ is selected from the group consisting of glutamic acid,        aspartic acid or asparagine;    -   X₄₂ is selected from the group consisting of alanine, ornithine,        lysine and arginine;    -   X₄₅ is tyrosine or phenylalanine;    -   R₆₂ is selected from the group consisting of AYRPSE (SEQ ID NO:        14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide        glycine-proline-glutamic acid, a tripeptide        valine-asparagine-glutamine, a dipeptide proline-glutamic acid,        a dipeptide asparagine-glutamine, glutamine, glutamic acid and        an N-terminal amine; and    -   R₅₃ is COOH or CONH₂. In one embodiment the insulin peptide is a        two chain insulin analog. In another embodiment the insulin        peptide is a single chain insulin analog wherein the carboxy        terminus of the B chain is linked to the amino terminus of the A        chain via a peptide linker. Any of the previous disclosed single        chain insulin analogs having activity at the insulin receptor        and known to those skilled in the art are encompassed by the        present disclosure.

In one embodiment the insulin peptide of the conjugate is a two chaininsulin wherein the A and B chains are linked by interchain disulfidebonds, wherein the A chain comprises the sequenceGIVEQCCX₈X₉ICSLYQLENYCX₂₁—R₅₃ (SEQ ID NO: 73) and the B chain comprisesa sequence R₆₂-X₂₅LCGAX₃₀LVDALYLVCGDX₄₂GFY (SEQ ID NO: 75), wherein

-   -   X₈ is histidine or threonine;    -   X₉ is serine, lysine, or alanine;    -   X₂₁ is alanine, glycine or asparagine;    -   X₂₅ is histidine or threonine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid;    -   X₄₂ is selected from the group consisting of alanine ornithine        and arginine; and R₅₃ is COOH or CONH₂;    -   R₆₂ is selected from the group consisting of FVNQ (SEQ ID NO:        12), a tripeptide valine-asparagine-glutamine, a dipeptide        asparagine-glutamine, glutamine, and an N-terminal amine; and    -   R₅₃ is COOH or CONH₂. In one embodiment the A chain comprises        the sequence GIVEQCCX₈X₉ICSLYQLENYCX₂₁—R₅₃ (SEQ ID NO: 73) and        the B chain comprises

the B chain sequence comprises the sequenceFVKQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 147), orFVNQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 148), wherein

X₈ is histidine or threonine;

-   -   X₉ is serine, lysine, or alanine;    -   X₂₁ is alanine, glycine or asparagine;

X₂₅ is selected from the group consisting of histidine and threonine;and

R₆₃ is selected from the group consisting of YTX₂₈KT (SEQ ID NO: 149),YTKPT (SEQ ID NO: 150), YTX₂₈K (SEQ ID NO: 152), YTKP (SEQ ID NO: 151),YTPK (SEQ ID NO: 70), YTX₂₈, YT, Y and a bond.

In one embodiment the A chain comprises the sequenceGIVEQCCX₈SICSLYQLENYCX₂₁-R₅₃ (SEQ ID NO: 153) orGIVEQCCTSICSLYQLENYCN-R₅₃ (SEQ ID NO: 1) and the B chain comprises thesequence FVKQX₂₅LCGSHLVEALYLVCGERGFFYTEKT (SEQ ID NO: 154),FVNQX₂₅LCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO: 155),FVNQX₂₅LCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 156) orFVNQX₂₅LCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 157) wherein

X₈ is histidine or threonine;

-   -   X₂₁ is alanine, glycine or asparagine; X₂₅ is selected from the        group consisting of histidine and threonine and R₅₃ is COOH or        CONH₂. In one embodiment the A chain comprises a sequence        GIVEQCCTSICSLYQLENYCN-R₅₃ (SEQ ID NO: 1) and said B chain        comprises a sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID        NO: 2) wherein R₅₃ is COOH or CONH₂.

In one embodiment the insulin peptide is a single chain insulin analog.In one embodiment the peptide linker joining the B and A chains isselected from the group consisting of SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQID NO: 158), SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 159),GAGSSSX₅₇X₅₈ (SEQ ID NO: 76), GYGSSSX₅₇X₅₈ (SEQ ID NO: 21) andGYGSSSX₅₇X₅₈APQT; (SEQ ID NO: 77), wherein X₅₇ and X₅₈ are independentlyarginine, lysine or ornithine. In one embodiment both X₅₇ and X₅₈ areindependently arginine. In one embodiment the peptide linking moietyjoining the insulin A and B chains to form a single chain insulin analogis a peptide sequence

consisting of GYGSSSRR (SEQ ID NO: 18) GAGSSSRR (SEQ ID NO: 22) orGAGSSSRRAPQT (SEQ ID NO: 23).

In accordance with one embodiment, the linker (L in the formula Q-L-Y)is a linking group or a bond that covalently links the insulin peptideto the NHR ligand. In one embodiment the NHR ligand is linked to theside chain of an amino acid at position B28 or B29 of the insulinpeptide. In one embodiment the amino acid at position B28 or B29 of theinsulin peptide is lysine and the NHR ligand is linked to the side chainof the lysine. In one embodiment the NHR ligand is linked to the insulinpeptide via the N-terminal alpha amine of the insulin A or B chain. Inone embodiment the NHR ligand is linked to the insulin peptide via anamid bond formed between an amino group of the insulin peptide and acarboxy group of the NHR ligand, optionally through a spacer moiety.

In one embodiment the linker (L in the formula Q-L-Y) is a linking groupwherein L is stable in vivo, hydrolyzable in vivo, or metastable invivo. In one embodiment L comprises an ether moiety, or an amide moiety,an ester moiety, an acid-labile moiety, a reduction-labile moiety, anenzyme-labile moiety, a hydrazone moiety, a disulfide moiety, or acathepsin-cleavable moiety.

Structure of the NHR Ligand (Q)

The NHR ligand of the FGF21 based conjugates is partly or whollynon-peptidic and is hydrophobic or lipophilic. In some embodiments, theNHR ligand has a molecular weight that is about 5000 daltons or less, orabout 4000 daltons or less, or about 3000 daltons or less, or about 2000daltons or less, or about 1750 daltons or less, or about 1500 daltons orless, or about 1250 daltons or less, or about 1000 daltons or less, orabout 750 daltons or less, or about 500 daltons or less, or about 250daltons or less. The structure of Q can be in accordance with any of theteachings disclosed herein.

In the embodiments described herein, Q is conjugated to L (e.g. when Lis a linking group) or Y (e.g. when L is a bond) at any position of Qthat is capable of reacting with Y or L. One skilled in the art couldreadily determine the position and means of conjugation in view ofgeneral knowledge and the disclosure provided herein.

In any of the embodiments described herein wherein Q comprises atetracyclic skeleton having three 6-membered rings joined to one5-membered ring or a variation thereof (e.g. a Q that acts at thevitamin D receptor), the carbon atoms of the skeleton are referred to byposition number, as shown below:

For example, a modification having a ketone at position-6 refers to thefollowing structure:

NHR Ligand that Acts on a Type I Nuclear Hormone Receptor

In some embodiments of the invention, the NHR ligand (Q) acts on a TypeI nuclear hormone receptor. In some embodiments, Q can have anystructure that permits or promotes agonist activity upon binding of theligand to a Type I nuclear hormone receptor, while in other embodimentsQ is an antagonist of the Type I nuclear hormone receptor.

In exemplary embodiments, Q comprises a structure as shown in Formula A:

wherein R¹ and R², when present, are independently moieties that permitor promote agonist or antagonist activity upon binding of the compoundof Formula A to the Type I nuclear hormone receptor; R³ and R⁴ areindependently moieties that permit or promote agonist or antagonistactivity upon binding of the compound of Formula A to the Type I nuclearhormone receptor; and each dashed line represents an optional doublebond. Formula A may further comprise one or more substituents at one ormore of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 16, 17, 18,and 19. Contemplated optional substituents include, but are not limitedto, OH, NH₂, ketone, and C₁-C₁₈ alkyl groups.

In some embodiments, Q comprises a structure of Formula A wherein

R¹ is present and is hydrogen, C₁-C₇ alkyl; (C₀₋C₃ alkyl)C(O)C₁-C₇alkyl, (C₀₋C₃ alkyl)C(O)aryl, or SO₃H;

R² is present and is hydrogen, halo, OH, or C₁-C₇ alkyl;

R³ is hydrogen, halo, OH, or C₁-C₇ alkyl;

R⁴ is hydrogen, (C₀₋C₈ alkyl)halo, C₁₋C₈ alkyl, C₂₋C₈ alkenyl, C₂₋₁₈alkynyl, heteroalkyl, (C₀₋C₈ alkyl)aryl, (C₀₋C₈ alkyl)heteroaryl, (C₀₋C₈alkyl)OC₁₋C₈ alkyl, (C₀₋C₈ alkyl)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)OC₂₋C₈alkynyl, (C₀₋C₈ alkyl)OH, (C₀₋C₈ alkyl)SH, (C₀₋C₈ alkyl)NR²⁴C₁₋C₈ alkyl,(C₀₋C₈ alkyl)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)NR²⁴C₂₋C₈ alkynyl, (C₀₋C₈alkyl)NR²⁴H₂, (C₀₋C₈ alkyl)C(O)C₁₋C₈ alkyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈alkenyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈ alkynyl, (C₀₋C₈ alkyl)C(O)H, (C₀₋C₈alkyl)C(O)aryl, (C₀₋C₈ alkyl)C(O)heteroaryl, (C₀₋C₈ alkyl)C(O)OC₁₋C₈alkyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₈ alkynyl,(C₀₋C₈ alkyl)C(O)OH, (C₀₋C₈ alkyl)C(O)O aryl, (C₀₋C₈ alkyl)C(O)Oheteroaryl, (C₀₋C₈ alkyl)OC(O)C₁₋C₈ alkyl, (C₀₋C₈ alkyl)OC(O)C₂₋C₈alkenyl, (C₀₋C₈ alkyl)OC(O)C₂₋C₁₈ alkynyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₁₋C₈alkyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₈alkynyl, (C₀₋C₈ alkyl)C(O)NR²⁴H₂, (C₀₋C₈ alkyl)C(O)NR²⁴aryl, (C₀₋C₈alkyl)C(O)NR²⁴heteroaryl, (C₀₋C₈ alkyl)NR²⁴C(O)C₁₋C₈ alkyl, (C₀₋C₈alkyl)NR²⁴C(O)C₂₋C₈ alkenyl, or (C₀₋C₈ alkyl)NR²⁴C(O)C₂₋C₈ alkynyl,(C₀₋C₈ alkyl)NR²⁴C(O)OH, (C₀₋C₈ alkyl)OC(O)OC₁₋C₈ alkyl, (C₀₋C₈alkyl)OC(O)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)OC(O)OC₂₋C₈ alkynyl, (C₀₋C₈alkyl)OC(O)OH, (C₀₋C₈ alkyl)OC(O)NR²⁴C₁₋C₈ alkyl, (C₀₋C₈alkyl)OC(O)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)OC(O)NR²⁴C₂₋C₈ alkynyl,(C₀₋C₈ alkyl)OC(O)NR²⁴H₂, (C₀₋C₈ alkyl)NR²⁴(O)OC₁₋C₈ alkyl, (C₀₋C₈alkyl)NR²⁴(O)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)NR²⁴(O)OC₂₋C₈ alkynyl, or(C₀₋C₈ alkyl)NR²⁴(O)OH; and,

R²⁴ is hydrogen or C₁₋C₇ alkyl.

In some embodiments, R¹ is hydrogen, propionate, acetate, benzoate, orsulfate; R² is hydrogen or methyl; R³ is hydrogen or methyl; and R⁴ isacetate, cypionate, hemisucciniate, enanthate, or propionate.

In embodiments wherein Q comprises a structure of Formula A, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula A that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula A and means of conjugation of Formula A to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula A is conjugated to L or Y at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ofFormula A. In some embodiments, Formula A is conjugated to L or Y atposition 1, 3, 6, 7, 12, 10, 13, 16, 17, or 19 of Formula A.

In some embodiments, Q acts at an estrogen receptor (e.g. ERα, ERβ). Insome embodiments, Q permits or promotes agonist activity at the estrogenreceptor, while in other embodiments Q is an antagonist of ER. Inexemplary embodiments, Q can have a structure of Formula B:

wherein R¹, R⁵ and R⁶ are moieties that permit or promote agonist orantagonist activity upon binding of the compound of Formula B to theestrogen receptor. In some embodiments, Formula B further comprises oneor more substituents at one or more of positions 1, 2, 4, 6, 7, 8, 9,11, 12, 14, 15, and 16 (e.g. a ketone at position-6).

In some embodiments when Q comprises a structure of Formula B, whereinR¹ is hydrogen, C₁-C₇ alkyl; (C₀₋C₃ alkyl)C(O)C₁-C₇ alkyl, (C₀₋C₃alkyl)C(O)aryl, or SO₃H;

R⁵ is hydrogen, (C₀₋C₈ alkyl)halo, C₁₋C₈ alkyl, C₂₋C₈ alkenyl, C₂₋₁₈alkynyl, heteroalkyl, (C₀₋C₈ alkyl)aryl, (C₀₋C₈ alkyl)heteroaryl, (C₀₋C₈alkyl)OC₁₋C₈ alkyl, (C₀₋C₈ alkyl)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)OC₂₋C₈alkynyl, (C₀₋C₈ alkyl)OH, (C₀₋C₈ alkyl)SH, (C₀₋C₈ alkyl)NR²⁴C₁₋C₈ alkyl,(C₀₋C₈ alkyl)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)NR²⁴C₂₋C₈ alkynyl, (C₀₋C₈alkyl)NR²⁴H₂, (C₀₋C₈ alkyl)C(O)C₁₋C₈ alkyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈alkenyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈ alkynyl, (C₀₋C₈ alkyl)C(O)H, (C₀₋C₈alkyl)C(O)aryl, (C₀₋C₈ alkyl)C(O)heteroaryl, (C₀₋C₈ alkyl)C(O)OC₁₋C₈alkyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₈ alkynyl,(C₀₋C₈ alkyl)C(O)OH, (C₀₋C₈ alkyl)C(O)O aryl, (C₀₋C₈ alkyl)C(O)Oheteroaryl, (C₀₋C₈ alkyl)OC(O)C₁₋C₈ alkyl, (C₀₋C₈ alkyl)OC(O)C₂₋C₈alkenyl, (C₀₋C₈ alkyl)OC(O)C₂₋C₁₈ alkynyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₁₋C₈alkyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₈alkynyl, (C₀₋C₈ alkyl)C(O)NR²⁴H₂, (C₀₋C₈ alkyl)C(O)NR²⁴aryl, (C₀₋C₈alkyl)C(O)NR²⁴heteroaryl, (C₀₋C₈ alkyl)NR²⁴C(O)C₁₋C₈ alkyl, (C₀₋C₈alkyl)NR²⁴C(O)C₂₋C₈ alkenyl, or (C₀₋C₈ alkyl)NR²⁴C(O)C₂₋C₈ alkynyl,(C₀₋C₈ alkyl)NR²⁴C(O)OH, (C₀₋C₈ alkyl)OC(O)OC₁₋C₈ alkyl, (C₀₋C₈alkyl)OC(O)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)OC(O)OC₂₋C₈ alkynyl, (C₀₋C₈alkyl)OC(O)OH, (C₀₋C₈ alkyl)OC(O)NR²⁴C₁₋C₈ alkyl, (C₀₋C₈alkyl)OC(O)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)OC(O)NR²⁴C₂₋C₈ alkynyl,(C₀₋C₈ alkyl)OC(O)NR²⁴H₂, (C₀₋C₈ alkyl)NR²⁴(O)OC₁₋C₈ alkyl, (C₀₋C₈alkyl)NR²⁴(O)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)NR²⁴(O)OC₂-C₈ alkynyl, or(C₀₋C₈ alkyl)NR²⁴(O)OH;

R⁶ is hydrogen, C₁₋C₈ alkyl, C₂₋C₈ alkenyl, C₂₋C₈ alkynyl, heteroalkyl,(C₀₋C₈ alkyl)aryl, (C₀₋C₈ alkyl)heteroaryl, (C₀₋C₈ alkyl)C(O)C₁₋C₈alkyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈ alkynyl,(C₀₋C₈ alkyl)C(O)H, (C₀₋C₈ alkyl)C(O)aryl, (C₀₋C₈ alkyl)C(O)heteroaryl,(C₀₋C₈ alkyl)C(O)OC₁₋C₈ alkyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₈ alkenyl, (C₀₋C₈alkyl)C(O)OC₂₋C₈ alkynyl, (C₀₋C₈ alkyl)C(O)OH, C₀₋C₈ alkyl)C(O)O aryl,(C₀₋C₈ alkyl)C(O)O heteroaryl, (C₀₋C₈ alkyl)C(O)NR²⁴C₁₋C₈ alkyl, (C₀₋C₈alkyl)C(O)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₈ alkynyl, (C₀₋C₈alkyl)C(O)NR²⁴H₂, (C₀₋C₈ alkyl)C(O)NR²⁴aryl, or (C₀₋C₈alkyl)C(O)NR²⁴heteroaryl; and

R²⁴ is hydrogen or C₁₋C₇ alkyl.

For example, R¹ is hydrogen, propionate, acetate, benzoate, or sulfate;R⁵ is hydrogen, ethynyl, hydroxyl; and R⁶ is acetate, cypionate,hemisucciniate, enanthate, or propionate.

Nonlimiting examples of the compound of Formula B include 17β-estradiol,modified forms of estradiol such as β-estradiol 17-acetate, β-estradiol17-cypionate, β-estradiol 17-enanthate, β-estradiol 17-valerate,β-estradiol 3,17-diacetate, β-estradiol 3,17-dipropionate, β-estradiol3-benzoate, β-estradiol 3-benzoate 17-n-butyrate, β-estradiol 3-glycidylether, β-estradiol 3-methyl ether, β-estradiol 6-one, β-estradiol3-glycidyl, β-estradiol 6-one 6-(O-carboxymethyloxime), 16-epiestriol,17-epiestriol, 2-methoxy estradiol, 4-methoxy estradiol, estradiol17-phenylpropionate, and 17β-estradiol 2-methyl ether,17α-ethynylestradiol, megestrol acetate, estriol, and derivativesthereof. In some embodiments, carbon 17 has a ketone substitutent and R⁵and R⁶ are absent (e.g. estrone). Some of the aforementioned compoundsof Formula B are shown below:

In embodiments wherein Q comprises a structure of Formula B, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula B that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula B and means of conjugation of Formula B to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula B is conjugated to L or Y at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ofFormula B. In some embodiments, Formula B is conjugated to L or Y atposition 3 or 17 of Formula B.

In other embodiments, Q acts at an estrogen receptor but is notencompassed by Formula B. Nonlimiting examples of ligands that act at anestrogen receptor that are not encompassed by Formula B are shown below:

In some embodiments, Q acts at a glucocorticoid receptor (GR). In someembodiments, Q comprises any structure that permits or promotes agonistactivity at the GR, while in other embodiments Q is an antagonist of GR.In exemplary embodiments, Q comprises a structure of Formula C:

wherein R², R³, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently moietiesthat permit or promote agonist or antagonist activity upon the bindingof the compound of Formula C to the GR; and each dash represents anoptional double bond. In some embodiments, Formula C further comprisesone or more substituents at one or more of positions 1, 2, 4, 5, 6, 7,8, 9, 11, 12, 14, and 15 (e.g. hydroxyl or ketone at position-11).

In some embodiments, Q comprises a structure of Formula C wherein

R² is hydrogen, halo, OH, or C₁-C₇ alkyl;

R³ is hydrogen, halo, OH, or C₁-C₇ alkyl;

R⁶ is hydrogen, C₁₋C₈ alkyl, C₂₋C₈ alkenyl, C₂₋C₈ alkynyl, heteroalkyl,(C₀₋C₈ alkyl)aryl, (C₀₋C₈ alkyl)heteroaryl, (C₀₋C₈ alkyl)C(O)C₁₋C₈alkyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈ alkynyl,(C₀₋C₈ alkyl)C(O)H, (C₀₋C₈ alkyl)C(O)aryl, (C₀₋C₈ alkyl)C(O)heteroaryl,(C₀₋C₈ alkyl)C(O)OC₁₋C₈ alkyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₈ alkenyl, (C₀₋C₈alkyl)C(O)OC₂₋C₈ alkynyl, (C₀₋C₈ alkyl)C(O)OH, C₀₋C₈ alkyl)C(O)O aryl,(C₀₋C₈ alkyl)C(O)O heteroaryl, (C₀₋C₈ alkyl)C(O)NR²⁴C₁₋C₈ alkyl, (C₀₋C₈alkyl)C(O)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₈ alkynyl, (C₀₋C₈alkyl)C(O)NR²⁴H₂, (C₀₋C₈ alkyl)C(O)NR²⁴aryl, or (C₀₋C₈alkyl)C(O)NR²⁴heteroaryl;

R⁷ is hydrogen, C₁₋C₈ alkyl, C₂₋C₈ alkenyl, C₂₋C₈ alkynyl, heteroalkyl,(C₀₋C₈ alkyl)aryl, (C₀₋C₈ alkyl)heteroaryl, (C₀ alkyl)C(O)C₁₋C₈ alkyl,(C₀ alkyl)C(O)C₂₋C₈ alkenyl, (C₀ alkyl)C(O)C₂₋C₈ alkynyl, (C₀)C(O)aryl,(C₀)C(O)heteroaryl, (C₀)C(O)OC₁₋C₈ alkyl, (C₀ alkyl)C(O)OC₂₋C₈ alkenyl,(C₀ alkyl)C(O)OC₂₋C₈ alkynyl, or (C₀ alkyl)C(O)OH;

R⁸ is hydrogen or C₁₋C₇ alkyl;

R⁹ is hydrogen or C₁₋C₇ alkyl;

R¹⁰ is hydrogen or OH; and

R²⁴ is hydrogen or C₁₋C₇ alkyl.

For example, R² is hydrogen or methyl; R³ is hydrogen, fluoro, chloro,or methyl; R⁶ is hydrogen or C(O) C₁-C₇ alkyl; R⁷ is hydrogen, C(O)CH₃,or C(O)CH₂CH₃; R⁸ is hydrogen or methyl; R⁹ is hydrogen or methyl; andR¹⁰ is hydroxyl.

Nonlimiting examples of structures of Formula C include:

and derivatives thereof.

In embodiments wherein Q comprises a structure of Formula C, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula C that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula C and means of conjugation of Formula C to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula C is conjugated to L or Y at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,or 23 of Formula C. In some embodiments, Formula C is conjugated to L orY at position 3, 10, 16 or 17 of Formula C.

In some embodiments, Q acts at a mineralcorticoid receptor (MR). In someembodiments, Q comprises any structure that permits or promotes agonistactivity at the MR, while in other embodiments Q is an antagonist of MR.In exemplary embodiments, Q comprises a structure of Formula D:

wherein R², R³, R⁷ and R¹⁰ are each independently a moiety that permitsor promotes agonist or antagonist activity upon binding of the compoundof Formula D to the MR; and the dashed line indicates an optional doublebond. In some embodiments, Formula D further comprises one or moresubstituents at one or more of positions 1, 2, 4, 5, 6, 7, 8, 11, 12,14, 15, 16, and 17.

In some embodiments, Q comprises a structure of Formula D wherein

R² is hydrogen, halo, OH, or C₁-C₇ alkyl;

R³ is hydrogen, halo, OH, or C₁-C₇ alkyl;

R⁷ is hydrogen, C₁₋C₈ alkyl, C₂₋C₈ alkenyl, C₂₋C₈ alkynyl, heteroalkyl,(C₀₋C₈ alkyl)aryl, (C₀₋C₈ alkyl)heteroaryl, (C₀ alkyl)C(O)C₁₋C₈ alkyl,(C₀ alkyl)C(O)C₂₋C₈ alkenyl, (C₀ alkyl)C(O)C₂₋C₈ alkynyl, (C₀)C(O)aryl,(C₀)C(O)heteroaryl, (C₀)C(O)OC₁₋C₈ alkyl, (C₀ alkyl)C(O)OC₂₋C₈ alkenyl,(C₀ alkyl)C(O)OC₂₋C₈ alkynyl, or (C₀ alkyl)C(O)OH;

R¹⁰ is hydrogen or OH; and

R²⁴ is hydrogen or C₁₋C₇ alkyl.

For example, R² is hydrogen or methyl; R³ is hydrogen, fluoro, chloro,or methyl; R⁷ is hydrogen, C(O)CH₃, or C(O)CH₂CH₃; and R¹⁰ is hydroxyl.

Nonlimiting examples of compounds of Formula D include:

and derivatives thereof.

In embodiments wherein Q comprises a structure of Formula D, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula D that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula D and means of conjugation of Formula D to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula D is conjugated to L or Y at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 of Formula D. In some embodiments, Formula D is conjugated toL or Y at position 3, 10, 13, or 17 of Formula D.

In some embodiments, Q acts at a progesterone receptor (PR). In someembodiments, Q comprises any structure that permits or promotes agonistactivity at the PR, while in other embodiments Q is an antagonist of PR.In exemplary embodiments, Q comprises a structure of Formula E:

wherein R², R³, R⁴, and R⁷ are each independently moieties that permitor promote agonist or antagonist activity upon binding of the compoundof Formula E to the PR; and the dashed line indicates an optional doublebond. In some embodiments, Formula E further comprises one or moresubstituents at one or more of positions 1, 2, 4, 5, 6, 7, 8, 11, 12,14, 15, 16, and 17 (e.g. a methyl group at position 6).

In some embodiments, Q comprises a structure of Formula E wherein

R² is hydrogen, halo, OH, or C₁-C₇ alkyl;

R³ is hydrogen, halo, OH, or C₁-C₇ alkyl;

R⁴ is hydrogen, (C₀₋C₈ alkyl)halo, C₁₋C₈ alkyl, C₂₋C₈ alkenyl, C₂₋₁₈alkynyl, heteroalkyl, (C₀₋C₈ alkyl)aryl, (C₀₋C₈ alkyl)heteroaryl, (C₀₋C₈alkyl)OC₁₋C₈ alkyl, (C₀₋C₈ alkyl)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)OC₂₋C₈alkynyl, (C₀₋C₈ alkyl)OH, (C₀₋C₈ alkyl)SH, (C₀₋C₈ alkyl)NR²⁴C₁₋C₈ alkyl,(C₀₋C₈ alkyl)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)NR²⁴C₂₋C₈ alkynyl, (C₀₋C₈alkyl)NR²⁴H₂, (C₀₋C₈ alkyl)C(O)C₁₋C₈ alkyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈alkenyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈ alkynyl, (C₀₋C₈ alkyl)C(O)H, (C₀₋C₈alkyl)C(O)aryl, (C₀₋C₈ alkyl)C(O)heteroaryl, (C₀₋C₈ alkyl)C(O)OC₁₋C₈alkyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₈ alkynyl,(C₀₋C₈ alkyl)C(O)OH, (C₀₋C₈ alkyl)C(O)O aryl, (C₀₋C₈ alkyl)C(O)Oheteroaryl, (C₀₋C₈ alkyl)OC(O)C₁₋C₈ alkyl, (C₀₋C₈ alkyl)OC(O)C₂₋C₈alkenyl, (C₀₋C₈ alkyl)OC(O)C₂₋C₁₈ alkynyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₁₋C₈alkyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₈alkynyl, (C₀₋C₈ alkyl)C(O)NR²⁴H₂, (C₀₋C₈ alkyl)C(O)NR²⁴aryl, (C₀₋C₈alkyl)C(O)NR²⁴heteroaryl, (C₀₋C₈ alkyl)NR²⁴C(O)C₁₋C₈ alkyl, (C₀₋C₈alkyl)NR²⁴C(O)C₂₋C₈ alkenyl, or (C₀₋C₈ alkyl)NR²⁴C(O)C₂₋C₈ alkynyl,(C₀₋C₈ alkyl)NR²⁴C(O)OH, (C₀₋C₈ alkyl)OC(O)OC₁₋C₈ alkyl, (C₀₋C₈alkyl)OC(O)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)OC(O)OC₂₋C₈ alkynyl, (C₀₋C₈alkyl)OC(O)OH, (C₀₋C₈ alkyl)OC(O)NR²⁴C₁₋C₈ alkyl, (C₀₋C₈alkyl)OC(O)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)OC(O)NR²⁴C₂₋C₈ alkynyl,(C₀₋C₈ alkyl)OC(O)NR²⁴H₂, (C₀₋C₈ alkyl)NR²⁴(O)OC₁₋C₈ alkyl, (C₀₋C₈alkyl)NR²⁴(O)OC₂₋C₈ alkenyl, (C₀₋C₈ alkyl)NR²⁴(O)OC₂-C₈ alkynyl, or(C₀₋C₈ alkyl)NR²⁴(O)OH;

R⁷ is hydrogen, C₁₋C₈ alkyl, C₂₋C₈ alkenyl, C₂₋C₈ alkynyl, heteroalkyl,(C₀₋C₈ alkyl)aryl, (C₀₋C₈ alkyl)heteroaryl, (C₀ alkyl)C(O)C₁₋C₈ alkyl,(C₀ alkyl)C(O)C₂₋C₈ alkenyl, (C₀ alkyl)C(O)C₂₋C₈ alkynyl, (C₀)C(O)aryl,(C₀)C(O)heteroaryl, (C₀)C(O)OC₁₋C₈ alkyl, (C₀ alkyl)C(O)OC₂₋C₈ alkenyl,(C₀ alkyl)C(O)OC₂₋C₈ alkynyl, or (C₀ alkyl)C(O)OH; and

R²⁴ is hydrogen or C₁₋C₇ alkyl.

For example, R² is hydrogen or methyl; R³ is hydrogen or methyl; R⁴ is(C₁ alkyl)C(O)C₁₋C₄ alkyl, acetate, cypionate, hemisucciniate,enanthate, or propionate; and R⁷ is hydrogen, C(O)CH₃, or C(O)CH₂CH₃

Nonlimiting examples of compounds of Formula E include:

and derivatives thereof.

In embodiments wherein Q comprises a structure of Formula E, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula E that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula E and means of conjugation of Formula E to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula E is conjugated to L or Y at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 of Formula E. In some embodiments, Formula E is conjugated toL or Y through position 3 or 17 of Formula E.

In other embodiments, Q acts at a progesterone receptor but is notencompassed by Formula E. For example, Q can comprise the belowstructure and analogs thereof:

In some embodiments, Q acts at an androgen receptor (AR). In someembodiments, Q comprises any structure that permits or promotes agonistactivity at the AR, while in other embodiments Q is an antagonist of AR.In exemplary embodiments, Q comprises a structure of Formula F:

wherein R¹, when present, R², R³ and R⁶ are each independently a moietythat permits or promotes agonist or antagonist activity upon binding ofthe compound of Formula F to the AR; and each dashed line represents anoptional double bond, with the proviso that no more than one of theoptional carbon-carbon double bond is present at position 5. In someembodiments, Formula F further comprises one or more substituents at oneor more of positions 1, 2, 4, 5, 6, 7, 8, 11, 12, 14, 15, 16, and 17.

In some embodiments, Q comprises a structure of Formula F wherein

R¹ is hydrogen, C₁-C₇ alkyl; (C₀₋C₃ alkyl)C(O)C₁-C₇ alkyl, (C₀₋C₃alkyl)C(O)aryl, or SO₃H;

R² is hydrogen, halo, OH, or C₁-C₇ alkyl;

R³ is hydrogen, halo, OH, or C₁-C₇ alkyl;

R⁶ is hydrogen, C₁₋C₈ alkyl, C₂₋C₈ alkenyl, C₂₋C₈ alkynyl, heteroalkyl,(C₀₋C₈ alkyl)aryl, (C₀₋C₈ alkyl)heteroaryl, (C₀₋C₈ alkyl)C(O)C₁₋C₈alkyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)C₂₋C₈ alkynyl,(C₀₋C₈ alkyl)C(O)H, (C₀₋C₈ alkyl)C(O)aryl, (C₀₋C₈ alkyl)C(O)heteroaryl,(C₀₋C₈ alkyl)C(O)OC₁₋C₈ alkyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₈ alkenyl, (C₀₋C₈alkyl)C(O)OC₂₋C₈ alkynyl, (C₀₋C₈ alkyl)C(O)OH, C₀₋C₈ alkyl)C(O)O aryl,(C₀₋C₈ alkyl)C(O)O heteroaryl, (C₀₋C₈ alkyl)C(O)NR²⁴C₁₋C₈ alkyl, (C₀₋C₈alkyl)C(O)NR²⁴C₂₋C₈ alkenyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₈ alkynyl, (C₀₋C₈alkyl)C(O)NR²⁴H₂, (C₀₋C₈ alkyl)C(O)NR²⁴aryl, or (C₀₋C₈alkyl)C(O)NR²⁴heteroaryl; and

R²⁴ is hydrogen or C₁₋C₇ alkyl.

For example, R¹ is hydrogen or absent; R² is hydrogen or methyl; R³ ishydrogen or methyl; and R⁶ is H or absent.

Nonlimiting examples of compounds of Formula F include:

and derivatives thereof.

In embodiments wherein Q comprises a structure of Formula F, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula F that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula F and means of conjugation of Formula F to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula F is conjugated to L or Y at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or22 of Formula F. In some embodiments, Formula F is conjugated to L or Yat position 3 or 17 of Formula F.

In some embodiments, the binding of the NHR ligand to the Type I nuclearhormone receptor results in agonist activity (or antagonist activity) insome but not all cells or tissues expressing the Type I nuclear hormonereceptor.

NHR Ligand that Acts on a Type II Nuclear Hormone Receptor

In some embodiments of the invention, the NHR ligand (Q) acts on a TypeII nuclear hormone receptor. In some embodiments, Q can have anystructure that permits or promotes agonist activity upon binding of theligand to a Type II nuclear hormone receptor, while in other embodimentsQ is an antagonist of the Type II nuclear hormone receptor. In exemplaryembodiments, Q exhibits agonist (or antagonist) activity at a thyroidhormone receptor (TR), retinoic acid receptor (RAR), peroxisomeproliferator activated receptor (PPAR), Liver X Receptor (LXR),farnesoid X receptor (FXR), vitamin D receptor (VDR), and/or pregnane Xreceptor (PXR).

In some embodiments, Q acts at a thyroid hormone receptor (e.g. TRα,TRβ). In some embodiments, Q comprises any structure that permits orpromotes agonist activity at the TR, while in other embodiments Q is anantagonist of TR. In one embodiment a thyroid hormone receptor agonistis provided having the general structure of

wherein

R₁₅ is C₁-C₄ alkyl, —CH₂(pyridazinone), —CH₂(OH)(phenyl)F, —CH(OH)CH₃,halo or H;

R₂₀ is halo, CH₃ or H;

R₂₁ is halo, CH₃ or H;

R₂₂ is H, OH, halo, —CH₂(OH)(C₆ aryl)F, or C₁-C₄ alkyl; and

R₂₃ is —CH₂CH(NH₂)COOH, —OCH₂COOH, —NHC(O)COOH, —CH₂COOH,

—NHC(O)CH₂COOH, —CH₂CH₂COOH, or —OCH₂PO₃ ²⁻.

In accordance with one embodiment the thyroid hormone receptor agonistis a compound of the general structure

wherein

R₁₅ is C₁-C₄ alkyl, —CH(OH)CH₃, I or H

R₂₀ is I, Br, CH₃ or H;

R₂₁ is I, Br, CH₃ or H;

R₂₂ is H, OH, I, or C₁-C₄ alkyl; and

R₂₃ is —CH₂CH(NH₂)COOH, —OCH₂COOH, —NHC(O)COOH, —CH₂COOH,

—NHC(O)CH₂COOH, —CH₂CH₂COOH, or —OCH₂PO₃ ²⁻. In one embodiment R₂₃ is—CH₂CH(NH₂)COOH.

In accordance with one embodiment the thyroid hormone receptor agonistis a compound of the general structure

wherein

R₁₅ is isopropyl, —CH(OH)CH₃, I or H

R₂₀ is I, Br, Cl, or CH₃;

R₂₁ is I, Br, Cl, or CH₃;

R₂₂ is H; and

R₂₃ is —OCH₂COOH, —CH₂COOH, —NHC(O)CH₂COOH, or —CH₂CH₂COOH.

In accordance with one embodiment the thyroid hormone receptor agonistis a compound of the general structure of Formula I:

wherein

R₂₀, R₂₁, and R₂₂ are independently selected from the group consistingof H, OH, halo and C₁-C₄ alkyl; and

R₁₅ is halo or H. In one embodiment R₂₀ and R₂₁ are each CH₃, R₁₅ is Hand R₂₂ are independently selected from the group consisting of H, OH,halo and C₁-C₄ alkyl. In one embodiment R₂₀, R₂₁ and R₂₂ are each haloand R₁₅ is H or halo. In a further embodiment R₂₀, R₂₁ and R₂₂ are eachI, and R₁₅ is H or I. In accordance with one embodiment Q is selectedfrom the group consisting of thyroxine T4 (3,5,3′,5′-tetraiodothyronine)and 3,5,3′-triiodo L-thyronine.

In one embodiment, the thyroid hormone receptor ligand (Y) of the Q-L-Yconjugates, is an indole derivative of thyroxine, including for example,compounds disclosed in U.S. Pat. No. 6,794,406 and US publishedapplication no. US 2009/0233979, the disclosures of which areincorporated herein. In one embodiment the indole derivative ofthyroxine comprises a compound of the general structure of Formula II:

wherein

R₁₃ is H or C₁-C₄ alkyl;

R₁₄ is C₁-C₈ alkyl;

R₁₅ is H or C₁-C₄ alkyl; and

R₁₆ and R₁₇ are independently halo or C₁-C₄ alkyl.

In one embodiment, the thyroid receptor ligand (Y) of the Q-L-Yconjugates, is an indole derivative of thyroxine as disclosed inWO97/21993 (U. Cal SF), WO99/00353 (KaroBio), GB98/284425 (KaroBio), andU.S. Provisional Application 60/183,223, the disclosures of which areincorporated by reference herein. In one embodiment the thyroid receptorligand comprises the general structure of Formula III:

wherein X is oxygen, sulfur, carbonyl, methylene, or NH;

Y is (CH₂)_(n), where n is an integer from 1 to 5, or C═C;

R₁ is halogen, trifluoromethyl, or C₁-C₆ alkyl or C₃-C₇ cycloalkyl;

R₂ and R₃ are the same or different and are hydrogen, halogen, C₁-C₆alkyl or C₃-C₇ cycloalkyl, with the proviso that at least one of R₂ andR₃ being other than hydrogen;

R₄ is hydrogen or C₁-C₄ alkyl;

R₅ is hydrogen or C₁-C₄ alkyl;

R₆ is carboxylic acid, or ester thereof;

R₇ is hydrogen, or an alkanoyl or aroyl group.

Nonlimiting examples of Q include the following compounds:

and derivatives thereof.

In embodiments wherein Q comprises a structure that permits or promotesagonist or antagonist activity at a TR, Q is conjugated to L (e.g. whenL is a linking group) or Y (e.g. when L is a bond) at any position of Qthat is capable of reacting with Y or L. One skilled in the art couldreadily determine the position of conjugation on Q and means ofconjugation of Q to Y or L in view of general knowledge and thedisclosure provided herein. In some embodiments, Q is conjugated to L orY through any position of Q. In some embodiments, Q is conjugated to Lor Y through the carboxylic acid or amine moieties, as indicated below:

In some embodiments, Q acts at a retinoic acid receptor (e.g. RARα,RARβ, RARγ). In some embodiments, Q comprises any structure that permitsor promotes agonist activity at the RAR, while in other embodiments Q isan antagonist of RAR. In exemplary embodiments, Q comprises a structureof Formula G:

wherein R¹¹ is a moiety that permits or promotes agonist or antagonistactivity upon the binding of the compound of Formula G to a RAR, and

represents either E or Z stereochemistry.

In some embodiments, Q comprises a structure of Formula G wherein R¹¹ isC(O)OH, CH₂OH, or C(O)H. In some embodiments, Q comprises a structure ofFormula G wherein R¹¹ is a carboxylic acid derivative (e.g. acylchloride, anhydride, and ester).

Nonlimiting examples of the compound of Formula G include:

and derivatives thereof.

In embodiments wherein Q comprises a structure of Formula G, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula G that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Q and means of conjugation of Q to Y or L in view ofgeneral knowledge and the disclosure provided herein. In someembodiments, Q is conjugated to L or Y through any position of Q. Insome embodiments, Formula G is conjugated to L or Y at R¹¹.

In some embodiments, Q acts at a peroxisome proliferator activatedreceptor (e.g. PPARα, PPARβ/δ, PPARγ). In some embodiments, Q acts atPPARγ. In some embodiments, Q comprises any structure that permits orpromotes agonist activity at the PPAR, while in other embodiments Q isan antagonist of PPAR. In some embodiments, Q is a saturated orunsaturated, halogenated or nonhalogenated free fatty acid (FFA) asdescribed by Formula H:

wherein n is 0-26 and each R¹², when present, is independently a moietythat permits or promotes agonist or antagonist activity upon binding ofthe compound of Formula H to a PPAR.

In some embodiments, Q comprises a structure of Formula H, wherein n is0-26 and each R¹², when present, is independently hydrogen, C₁-C₇ alkyl,or halogen. In some embodiments Formula B is saturated such as, forexample, formic acid, acetic acid, n-caproic acid, heptanoic acid,caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid,tridecanoic acid, myristic acid, pentadeconoic acid, palmitic acid,heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid,heneicosanoic acid, behenic acid, tricosanoic acid, perfluorononanoicacid (see below), perfluorooctanoic acid (see below), and derivativesthereof. In some embodiments Formula H is unsaturated with either cis ortrans stereochemistry such as, for example, mead acid, myristoleic acid,palmitoleic acid, sapienic acid, oleic acid, linoleic acid, α-linolenicacid, elaidic acid, petroselinic acid, arachidonic acid,dihydroxyeicosatetraenoic acid (DiHETE), octadecynoic acid,eicosatriynoic acid, eicosadienoic acid, eicosatrienoic acid,eicosapentaenoic acid, erucic acid, dihomolinolenic acid, docosatrienoicacid, docosapentaenoic acid, docosahexaenoic acid, adrenic acid, andderivatives thereof, including for example:

In embodiments wherein Q comprises a structure of Formula H, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula H that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula H and means of conjugation of Formula H to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula H is conjugated to L or Y at any position onFormula H. In some embodiments, Formula H is conjugated to L or Ythrough the terminal carboxylic acid moiety.

In some of these embodiments, Q is an eiconsanoid. In specificembodiments, Q is a prostaglandin or a leukotriene. In some exemplaryembodiments, Q is a prostaglandin having a structure as described byFormula J1-J6:

wherein each R¹³ is independently a moiety that permits or promotesagonist or antagonist activity upon the binding of the compound ofFormula J to a PPAR (e.g. PGJ2 as shown below):

In some embodiments when Q comprises a structure of any one of FormulaJ146, each R¹³ is independently C₇-C₈ alkyl, C₇-C₈ alkenyl, C₇-C₈alkynyl, or heteroalkyl.

In embodiments wherein Q is an eicosanoid, Q is conjugated to L (e.g.when L is a linking group) or Y (e.g. when L is a bond) at any positionof the eicosanoid that is capable of reacting with Y or L. One skilledin the art could readily determine the position of conjugation on Q andmeans of conjugation of Q to Y or L in view of general knowledge and thedisclosure provided herein. In some embodiments, Q is conjugated to L orY through any position of Q. In some embodiments, the eicosanoid isconjugated to L or Y through a terminal carboxylic acid moiety orthrough a pendant alcohol moiety.

In some exemplary embodiments, Q is a leukotriene having a structure asdescribed by Formula K or a derivatized form of Formula K:

wherein each R¹⁴ is independently a moiety that permits or promotesagonist or antagonist activity upon the binding of the compound ofFormula K to a PPAR (e.g. leukotriene B4 as shown below):

In some embodiments when Q comprises a structure of Formula K, each R¹⁴is independently C₃-C₁₃ alkyl, C₃-C₁₃ alkenyl, C₃-C₁₃ alkynyl, orheteroalkyl.

In embodiments wherein Q comprises a structure of Formula K, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula K that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula K and means of conjugation of Formula K to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula K is conjugated to L or Y at any position onFormula K. In some embodiments, Formula K is conjugated to L or Ythrough the terminal carboxylic acid moiety or through a pendant alcoholmoiety.

In some exemplary embodiments, Q is a thiazolidinedione comprising astructure as described by Formula L:

Nonlimiting examples of the compound of Formula L include:

and derivatives thereof.

In embodiments wherein Q comprises a structure of Formula L, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula L that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula L and means of conjugation of Formula L to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula L is conjugated to L or Y at any position onFormula L, such as, for example, a pendant alcohol moiety, or through anaromatic substituent.

In one embodiment wherein Y is Tesaglitzar or Aleglitazar:

In embodiments wherein Q comprises Tesaglitzar or Aleglitazar, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position that is capable of reacting with Y or L. In oneembodiment, Tesaglitzar or Aleglitazar is conjugated to L or Y throughthe carboxylic acid moiety of the compound.

In some embodiments, Q acts at a RAR-related orphan receptor (e.g. RORα,RORβ, RORγ). In some embodiments, Q comprises any structure that permitsor promotes agonist activity at the ROR, while in other embodiments Q isan antagonist of ROR.

Nonlimiting examples of Q include:

and derivatives thereof.

In embodiments wherein Q acts at a ROR, Q is conjugated to L (e.g. whenL is a linking group) or Y (e.g. when L is a bond) at any position of Qthat is capable of reacting with Y or L. One skilled in the art couldreadily determine the position of conjugation on Y and means ofconjugation of Q to Y or L in view of general knowledge and thedisclosure provided herein. In some embodiments, Q is conjugated to L orY through any position of Q, such as, for example, any of the positionspreviously described herein.

In some embodiments, Q acts at a liver X receptor (LXRα, LXRβ). In someembodiments, Q comprises any structure that permits or promotes agonistactivity at the LXR, while in other embodiments Q is an antagonist ofLXR. In exemplary embodiments, Q is an oxysterol (i.e. oxygenatedderivative of cholesterol). Nonlimiting examples of Q in theseembodiments include 22(R)-hydroxycholesterol (see below),24(S)-hydroxycholesterol (see below), 27-hydroxycholesterol,cholestenoic acid, and derivatives thereof.

In embodiments wherein Q acts at a LXR, Q is conjugated to L (e.g. whenL is a linking group) or Y (e.g. when L is a bond) at any position of Ythat is capable of reacting with Y or L. One skilled in the art couldreadily determine the position of conjugation on Y and means ofconjugation of Q to Y or L in view of general knowledge and thedisclosure provided herein. In some embodiments, Q is conjugated to L orY at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 of Formula F. In someembodiments, Formula F is conjugated to L or Y at position 3 or 17 ofFormula F.

In some embodiments, Q acts at the farnesoid X receptor (FXR). In someembodiments, Q comprises any structure that permits or promotes agonistactivity at the FXR, while in other embodiments Q is an antagonist ofFXR. In some of these embodiments, Q is a bile acid. In exemplaryembodiments, Q has a structure of Formula M:

wherein each of R¹⁵, R¹⁶, and R¹⁷ are independently moieties that permitor promote agonist or antagonist activity upon binding of the compoundof Formula M to a FXR.

In some embodiments when Q comprises a structure of Formula M, each ofR¹⁵ and R¹⁶ are independently hydrogen, (C₀₋C₈ alkyl)halo, C₁₋C₁₈ alkyl,C₂₋C₁₈ alkenyl, C₂₋C₁₈ alkynyl, heteroalkyl, or (C₀₋C₈ alkyl)OH; and R¹⁷is OH, (C₀-C₈ alkyl)NH(C₁-C₄ alkyl)SO₃H, or (C₀-C₈ alkyl)NH(C₁-C₄alkyl)COOH.

In some embodiments when Q comprises a structure of Formula M, each ofR¹⁵ and R¹⁶ are independently hydrogen or OH; and R¹⁷ is OH, NH(C₁-C₂alkyl)SO₃H, or NH(C₁-C₂ alkyl)COOH.

Nonlimiting examples of the compound of Formula M include:

and derivatives thereof.

In embodiments wherein Q comprises a structure of Formula M, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula M that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula M and means of conjugation of Formula M to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula M is conjugated to L or Y at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 of Formula M. In some embodiments, Formula M is conjugatedto L or Y at position 3, 7, 12 or 17 of Formula M.

In some embodiments, Q acts at the vitamin D receptor (VDR). In someembodiments, Q comprises any structure that permits or promotes agonistactivity at the VDR, while in other embodiments Q is an antagonist ofVDR. In exemplary embodiments, Q has a structure of Formula N:

wherein each of R¹⁸, R¹⁹, R²⁰, R²¹, R²², and R²³ are moieties thatpermit or promote agonist or antagonist activity upon binding of thecompound of Formula N to the VDR such as, for example, any of thevitamin D compounds found in Bouillon et al., Endocrine Reviews,16(2):200-257 (1995).

In some embodiments wherein Q comprises a structure of Formula N,

R¹⁸ and R¹⁹ are each independently hydrogen, (C₀₋C₈ alkyl)halo, (C₀₋C₈alkyl)heteroaryl, or (C₀₋C₈ alkyl)OH;

both of R²⁰ are hydrogen or both of R²⁰ are taken together to form ═CH₂;

each of R²¹ and R²² are independently C₁-C₄ alkyl; and

R²³ is C₄₋C₁₈ alkyl, C₄₋C₁₈ alkenyl, C₄₋C₁₈ alkynyl, heteroalkyl,(C₄₋C₁₈ alkyl)aryl, (C₄₋C₁₈ alkyl)heteroaryl, (C₀₋C₈ alkyl)OC₁₋C₁₈alkyl, (C₀₋C₈ alkenyl)OC₁₋C₁₈ alkyl, (C₀₋C₈ alkynyl)OC₁₋C₁₈ alkyl,(C₀₋C₈ alkyl)OC₂₋C₁₈ alkenyl, (C₀₋C₈ alkyl)OC₂₋C₁₈ alkynyl, (C₆₋C₁₈alkyl)OH, (C₆₋C₁₈ alkyl)SH, (C₆₋C₁₈ alkenyl)OH, (C₆₋C₁₈ alkynyl)OH,(C₀₋C₈ alkyl)NR²⁴C₁-C₁₈ alkyl, (C₀₋C₈ alkenyl)NR²⁴C₁₋C₁₈ alkyl, (C₀₋C₈alkynyl)NR²⁴C₁₋C₁₈ alkyl, (C₀₋C₈ alkyl)NR²⁴C₂₋C₁₈ alkenyl, (C₀₋C₈alkyl)NR²⁴C₂₋C₁₈ alkynyl, (C₀₋C₈ alkyl)C(O)C₁₋C₁₈ alkyl, (C₀₋C₈alkyl)C(O)C₂₋C₁₈ alkenyl, (C₀₋C₈ alkyl)C(O)C₂₋C₁₈ alkynyl, (C₀₋C₈alkyl)C(O)H, (C₀₋C₈ alkyl)C(O)aryl, (C₀₋C₈ alkyl)C(O)heteroaryl, (C₀₋C₈alkyl)C(O)OC₁₋C₁₈ alkyl, (C₀₋C₈ alkyl)C(O)OC₂₋C₁₈ alkenyl, (C₀₋C₈alkyl)C(O)OC₂₋C₁₈ alkynyl, (C₀₋C₈ alkyl)C(O)OH, (C₀₋C₈ alkyl)C(O)O aryl,(C₀₋C₈ alkyl)C(O)O heteroaryl, (C₀₋C₈ alkyl)OC(O)C₁₋C₁₈ alkyl, (C₀₋C₈alkyl)OC(O)C₂₋C₁₈ alkenyl, (C₀₋C₈ alkyl)OC(O)C₂₋C₁₈ alkynyl, (C₀₋C₈alkyl)C(O)NR²⁴C₁-C₁₈ alkyl, (C₀₋C₈ alkyl)C(O)NR²⁴C₂₋C₁₈ alkenyl, (C₀₋C₈alkyl)C(O)NR²⁴C₂₋C₁₈ alkynyl, (C₀₋C₈ alkyl)C(O)NR²⁴H₂, (C₀₋C₈alkyl)C(O)NR²⁴aryl, (C₀₋C₈ alkyl)C(O)NR²⁴heteroaryl, (C₀₋C₈alkyl)NR²⁴C(O)C₁₋C₁₈ alkyl, (C₀₋C₈ alkyl)NR²⁴C(O)C₂₋C₈ alkenyl, or(C₀₋C₈ alkyl)NR²⁴C(O)C₂₋C₁₈ alkynyl, (C₀₋C₈ alkyl)NR²⁴C(O)OH, (C₀₋C₈alkyl)OC(O)OC₁₋C₁₈ alkyl, (C₀₋C₈ alkyl)OC(O)OC₂₋C₁₈ alkenyl, (C₀₋C₈alkyl)OC(O)OC₂₋C₁₈ alkynyl, (C₀₋C₈ alkyl)OC(O)OH, (C₀₋C₈alkyl)OC(O)NR²⁴C₁₋C₁₈ alkyl, (C₀₋C₈ alkyl)OC(O)NR²⁴C₂₋C₁₈ alkenyl,(C₀₋C₈ alkyl)OC(O)NR²⁴C₂₋C₁₈ alkynyl, (C₀₋C₈ alkyl)OC(O)NR²⁴H₂, (C₀₋C₈alkyl)NR²⁴(O)OC₁₋C₁₈ alkyl, (C₀₋C₈ alkyl)NR²⁴(O)OC₂₋C₁₈ alkenyl, (C₀₋C₈alkyl)NR²⁴(O)OC₂₋C₁₈ alkynyl, or (C₀₋C₈ alkyl)NR²⁴(O)OH; and

R²⁴ is hydrogen or C₁₋C₁₈ alkyl.

Nonlimiting examples of the compound of Formula N include:

and derivatives thereof.

In embodiments wherein Q comprises a structure of Formula N, Q isconjugated to L (e.g. when L is a linking group) or Y (e.g. when L is abond) at any position of Formula N that is capable of reacting with Y orL. One skilled in the art could readily determine the position ofconjugation on Formula N and means of conjugation of Formula N to Y or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula N is conjugated to L or Y at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or 26 of Formula N. In some embodiments, Formula N isconjugated to L or Y at position 1, 3, 19, or 25 of Formula N.

In some embodiments, Q acts at the pregnane X receptor (PXR). In someembodiments, Q comprises any structure that permits or promotes agonistactivity at the PXR, while in other embodiments Q is an antagonist ofPXR. In some embodiments, Q is a steroid, antibiotic, antimycotic, bileacid, hyperforin, or a herbal compound. In exemplary embodiments, Q iscompound that is able to induce CYP3A4, such as dexamethasone andrifampicin. In embodiments wherein Q comprises a structure that acts atthe PXR, Q is conjugated to L (e.g. when L is a linking group) or Y(e.g. when L is a bond) at any position of Q that is capable of reactingwith Y or L. One skilled in the art could readily determine the positionof conjugation on Y and means of conjugation of Q to Y or L in view ofgeneral knowledge and the disclosure provided herein. In someembodiments, Q is conjugated to L or Y at any of positions on Q.

Modification of the NHR Ligand (O)

In some embodiments, the NHR ligand is derivatized or otherwisechemically modified to comprise a reactive moiety that is capable ofreacting with the insulin peptide (Y) or the linking group (L). In theembodiments described herein, Q is derivatized at any position of Q thatis capable of reacting with Y or L. The position of derivatization on Qis apparent to one skilled in the art and depends on the type of NHRligand used and the activity that is desired. For example, inembodiments wherein Q has a structure comprising a tetracyclic skeletonhaving three 6-membered rings joined to one 5-membered ring or avariation thereof, Q can be derivatized at any of positions 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25. Other positions of derivatization can be as previouslydescribed herein.

The NHR ligand can be derivatized using any agent known to one skilledin the art or described herein. For example, estradiol can bederivatized with succinic acid, succinic anhydride, benzoic acid, ethyl2-bromoacetate, or iodoacetic acid to form the below derivatives ofestradiol that are capable of conjugating to Q or L.

Similarly, any of the aforementioned NHR ligands can be derivatized bymethods known in the art. Additionally, certain derivatized ligands arecommercially available and can be purchased from chemical companies suchas Sigma-Aldrich.

In accordance with one embodiment Q is selected from the groupconsisting of estradiol and derivatives thereof, estrone and derivativesthereof, testosterone and derivatives thereof, and cortisol andderivatives thereof. In one embodiment Q is dexamethasone. In oneembodiment Q is selected from the group consisting of thyroxine T4(3,5,3′,5′-tetra-iodothyronine), 3,5,3′-triiodo L-thyronine,Tesaglitazar, Aleglitazar and thiazolidinediones. In one embodiment Q isselected from the group consisting of thyroxine T4(3,5,3′,5′-tetra-iodothyronine), and 3,5,3′-triiodo L-thyronine. In oneembodiment Q is selected from the group consisting of Tesaglitazar andAleglitazar.

Structure of the Insulin Peptide

In some embodiments, the insulin peptide of the presently disclosedconjugates is native insulin, comprising the A chain of SEQ ID NO: 1 andthe B chain of SEQ ID NO: 2, or an analog of native insulin, includingfor example a single-chain insulin analog comprising SEQ ID NOS: 1 and2. In accordance with the present disclosure analogs of insulinencompass polypeptides comprising an A chain and a B chain wherein theinsulin analogs differ from native insulin by one or more amino acidsubstitutions at positions selected from A5, A8, A9, A10, A12, A14, A15,A17, A18, A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B17, B20, B21,B22, B23, B26, B27, B28, B29 and B30 or deletions of any or all ofpositions B1-4 and B26-30.

In one embodiment the insulin peptide is an insulin analog wherein:

-   -   (a) the amino acid residue at position B28 is substituted with        Asp, Lys, Leu, Val, or Ala, and the amino acyl residue at        position B29 is Lys or Pro;    -   (b) the amino acid residues at any of positions B27, B28, B29,        and B30 are deleted or substituted with a nonnative amino acid.        In one embodiment an insulin analog is provided comprising an        Asp substituted at position B28 or a Lys substituted at position        28 and a proline substituted at position B29. Additional insulin        analogs are disclosed in Chance, et al., U.S. Pat. No.        5,514,646; Chance, et al., U.S. patent application Ser. No.        08/255,297; Brems, et al., Protein Engineering, 5:527-533        (1992); Brange, et al., EPO Publication No. 214,826 (published        Mar. 18, 1987); and Brange, et al., Current Opinion in        Structural Biology, 1:934-940 (1991). The disclosures of which        are expressly incorporated herein by reference.

Insulin analogs may also have replacements of the amidated amino acidswith acidic forms. For example, Asn may be replaced with Asp or Glu.Likewise, Gln may be replaced with Asp or Glu. In particular, Asn(A18),Asn(A21), or Asp(B3), or any combination of those residues, may bereplaced by Asp or Glu. Also, Gln(A15) or Gln(B4), or both, may bereplaced by either Asp or Glu.

As disclosed herein single chain insulin agonists are providedcomprising a B chain and an A chain of human insulin, or analogs orderivative thereof, wherein the carboxy terminus of the B chain islinked to the amino terminus of the A chain via a linking moiety. In oneembodiment the A chain is an amino acid sequence selected from the groupconsisting of GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1),GIVDECCFRSCDLRRLEMYCA (SEQ ID NO: 5) or GIVEECCFRSCDLALLETYCA (SEQ IDNO: 7) and the B chain comprises the sequenceFVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2),GPETLCGAELVDALYLVCGDRGFYFNKPT (SEQ ID NO: 6) orAYRPSETLCGGELVDTLYLVCGDRGFYFSRPA (SEQ ID NO: 8), or a carboxy shortenedsequence thereof having one to five amino acids corresponding to B26,B27, B28, B29 and B30 deleted, and analogs of those sequences whereineach sequence is modified to comprise one to five amino acidsubstitutions at positions corresponding to native insulin positions(see peptide alignment shown in FIG. 5) selected from A5, A8, A9, A10,A14, A15, A17, A18, A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B20,B22, B23, B26, B27, B28, B29 and B30. In one embodiment the amino acidsubstitutions are conservative amino acid substitutions. Suitable aminoacid substitutions at these positions that do not adversely impactinsulin's desired activities are known to those skilled in the art, asdemonstrated, for example, in Mayer, et al., Insulin Structure andFunction, Biopolymers. 2007; 88(5):687-713, the disclosure of which isincorporated herein by reference.

Additional amino acid sequences can be added to the amino terminus ofthe B chain or to the carboxy terminus of the A chain of the singlechain insulin agonists of the present invention. For example, a seriesof negatively charged amino acids can be added to the amino terminus ofthe B chain, including for example a peptide of 1 to 12, 1 to 10, 1 to 8or 1 to 6 amino acids in length and comprising one or more negativelycharged amino acids including for example glutamic acid and asparticacid. In one embodiment the B chain amino terminal extension comprises 1to 6 charged amino acids. In one embodiment the B chain amino terminalextension comprises the sequence GX₆₁X₆₂X₆₃X₆₄X₆₅K (SEQ ID NO: 26) orX₆₁X₆₂X₆₃X₆₄X₆₅RK (SEQ ID NO: 27), wherein X₆₁, X₆₂, X₆₃ X₆₄ and X₆₅ areindependently glutamic acid or aspartic acid. In one embodiment the Bchain comprises the sequence GEEEEEKGPEHLCGAHLVDALYLVCGDX₄₂GFY (SEQ IDNO: 28), wherein X₄₂ is selected from the group consisting of alaninelysine, ornithine and arginine.

High potencyFGF21 based insulin conjugates can also be prepared based onusing a modified IGF I and IGF II sequence described in publishedInternational application no. WO 2010/080607, the disclosure of which isexpressly incorporated herein by reference, as the insulin peptidecomponent. More particularly, analogs of IGF I and IGF II that comprisea substitution of a tyrosine leucine dipeptide for the native IGF aminoacids at positions corresponding to B16 and B17 of native insulin have atenfold increase in potency at the insulin receptor.

In accordance with one embodiment the insulin peptide for use in thepresent disclosure comprises a B chain sequence ofR₆₂-X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGX₄₁X₄₂GFX₄₅ (SEQ ID NO: 20) and an A chainsequence of GIVX₄X₅CCX₈X₉X₁₀CX₁₂LX₁₄X₁₅LX₁₇X₁₈X₁₉CX₂₁—R₅₃ (SEQ ID NO:29) wherein

-   -   X₄ is glutamic acid or aspartic acid;    -   X₅ is glutamine or glutamic acid    -   X₈ is histidine, threonine or phenylalanine;    -   X₉ is serine, arginine, lysine, ornithine or alanine;    -   X₁₀ is isoleucine or serine;    -   X₁₂ is serine or aspartic acid    -   X₁₄ is tyrosine, arginine, lysine, ornithine or alanine;    -   X₁₅ is glutamine, glutamic acid, arginine, alanine, lysine,        ornithine or leucine;    -   X₁₇ is glutamine, glutamic acid, arginine, aspartic acid or        lysine, ornithine    -   X₁₈ is methionine, asparagine, glutamine, aspartic acid,        glutamic acid or threonine;    -   X₁₉ is tyrosine, 4-methoxy-phenylalanine or 4-amino        phenylalanine;    -   X₂₁ is selected from the group consisting of alanine, glycine,        serine, valine, threonine, isoleucine, leucine, glutamine,        glutamic acid, asparagine, aspartic acid, histidine, tryptophan,        tyrosine, and methionine;    -   X₂₅ is histidine or threonine;    -   X₂₉ is selected from the group consisting of alanine, glycine        and serine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid;    -   X₃₃ is selected from the group consisting of aspartic acid,        glutamine and glutamic acid;    -   X₃₄ is selected from the group consisting of alanine and        threonine;    -   X₄₁ is selected from the group consisting of glutamic acid,        aspartic acid or asparagine;    -   X₄₂ is selected from the group consisting of alanine, lysine,        ornithine and arginine;    -   X₄₅ is tyrosine, histidine, asparagine or phenylalanine;    -   R₆₂ is selected from the group consisting of AYRPSE (SEQ ID NO:        14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide        glycine-proline-glutamic acid, a tripeptide        valine-asparagine-glutamine, a dipeptide proline-glutamic acid,        a dipeptide asparagine-glutamine, glutamine, glutamic acid and a        bond; and R₅₃ is COOH or CONH₂. In one embodiment the A chain        and the B chain are linked to one another by interchain        disulfide bonds, including those that form between the A and B        chains of native insulin. In an alternative embodiment the A and        B chains are linked together as a linear single chain-insulin        peptide.

In one embodiment the conjugates comprise an insulin peptide wherein theA chain comprises a sequence of GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 30)and said B chain sequence comprises a sequence ofX₄LCGX₅X₆LVEALYLVCGERGFF (SEQ ID NO: 31), wherein

-   -   X₁ is selected from the group consisting of threonine and        histidine;    -   X₂ is tyrosine, 4-methoxy-phenylalanine or 4-amino        phenylalanine;    -   X₃ is selected from the group consisting of asparagine and        glycine;    -   X₄ is selected from the group consisting of histidine and        threonine;    -   X₅ is selected from the group consisting of alanine, glycine and        serine;    -   X₆ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid.

In accordance with one embodiment an insulin analog is provided whereinthe A chain of the insulin peptide comprises the sequenceGIVEQCCX₈X₉ICSLYQLENYCX₂₁—R₅₃ (SEQ ID NO: 73) orGIVEQCCX₈SICSLYQLX₁₇NYCX₂₁ (SEQ ID NO: 32) and the B chain comprisingthe sequence R₆₂-X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGX₄₁X₄₂GFX₄₅YT-Z₁-B₁ (SEQ IDNO: 142), wherein

-   -   X₈ is selected from the group consisting of threonine and        histidine;    -   X₉ is valine or tyrosine;    -   X₁₇ is glutamine or glutamic acid;    -   X₂₁ is asparagine or glycine;    -   X₂₅ is histidine or threonine;    -   X₂₉ is selected from the group consisting of alanine, glycine        and serine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid;    -   X₃₃ is selected from the group consisting of aspartic acid and        glutamic acid;    -   X₃₄ is selected from the group consisting of alanine and        threonine;    -   X₄₁ is selected from the group consisting of glutamic acid,        aspartic acid or asparagine;    -   X₄₂ is selected from the group consisting of alanine, ornithine,        lysine and arginine;    -   X₄₅ is tyrosine or phenylalanine;    -   R₆₂ is selected from the group consisting of FVNQ (SEQ ID NO:        12), a tripeptide valine-asparagine-glutamine, a dipeptide        asparagine-glutamine, glutamine and an N-terminal amine    -   Z₁ is a dipeptide selected from the group consisting of        aspartate-lysine, lysine-proline, and proline-lysine; and    -   B₁ is selected from the group consisting of threonine, alanine        or a threonine-arginine-arginine tripeptide.

In accordance with one embodiment an insulin analog is provided whereinthe A chain of the insulin peptide comprises the sequenceGIVEQCCX₈SICSLYQLX₁₇NX₁₉CX₂₁ (SEQ ID NO: 32) and the B chain comprisingthe sequence X₂₅LCGX₂₉X₃₀LVEALYLVCGERGFF (SEQ ID NO: 33) wherein

-   -   X₈ is selected from the group consisting of threonine and        histidine;    -   X₁₇ is glutamic acid or glutamine;    -   X₁₉ is tyrosine, 4-methoxy-phenylalanine or 4-amino        phenylalanine;    -   X₂₁ is asparagine or glycine;    -   X₂₅ is selected from the group consisting of histidine and        threonine;    -   X₂₉ is selected from the group consisting of alanine, glycine        and serine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid. In a        further embodiment the B chain comprises the sequence        X₂₂VNQX₂₅LCGX₂₉X₃₀LVEALYLVCGERGFFYT-Z₁-B₁ (SEQ ID NO: 34)        wherein    -   X₂₂ is selected from the group consisting of phenylalanine and        desamino-phenylalanine;    -   X₂₅ is selected from the group consisting of histidine and        threonine;    -   X₂₉ is selected from the group consisting of alanine, glycine        and serine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid;    -   Z₁ is a dipeptide selected from the group consisting of        aspartate-lysine, lysine-proline, and proline-lysine; and    -   B₁ is selected from the group consisting of threonine, alanine        or a threonine-arginine-arginine tripeptide.

In accordance with some embodiments the A chain comprises the sequenceGIVEQCCX₈SICSLYQLX₁₇NX₁₉CX₂₃ (SEQ ID NO: 32) orGIVDECCX₈X₉SCDLX₁₄X₁₅LX₁₇X₁₈ X₁₉CX₂₁-R₅₃ (SEQ ID NO: 35), and the Bchain comprises the sequence X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGDX₄₂GFX₄₅ (SEQ IDNO: 36) wherein

-   -   X₈ is histidine or phenylalanine;    -   X₉ and X₁₄ are independently selected from arginine, lysine,        ornithine or alanine;    -   X₁₅ is arginine, lysine, ornithine or leucine;    -   X₁₇ is glutamic acid or glutamine;    -   X₁₈ is methionine, asparagine or threonine;    -   X₁₉ is tyrosine, 4-methoxy-phenylalanine or 4-amino        phenylalanine;    -   X₂₁ is alanine, glycine or asparagine;    -   X₂₃ is asparagine or glycine;    -   X₂₅ is selected from the group consisting of histidine and        threonine;    -   X₂₉ is selected from the group consisting of alanine, glycine        and serine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid;    -   X₃₃ is selected from the group consisting of aspartic acid and        glutamic acid;    -   X₃₄ is selected from the group consisting of alanine and        threonine;    -   X₄₂ is selected from the group consisting of alanine, lysine,        ornithine and arginine;    -   X₄₅ is tyrosine; and    -   R₅₃ is COOH or CONH₂.

In a further embodiment the A chain comprises the sequenceGIVDECCX₈X₉SCDLX₁₄X₁₅LX₁₇X₁₈ X₁₉CX₂₁—R₅₃ (SEQ ID NO: 35), and the Bchain comprises the sequence X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGDX₄₂GFX₄₅ (SEQ IDNO: 36) wherein

-   -   X₈ is histidine;    -   X₉ and X₁₄ are independently selected from arginine, lysine,        ornithine or alanine;    -   X₁₅ is arginine, lysine, ornithine or leucine;    -   X₁₇ is glutamic acid, aspartic acid, asparagine, lysine,        ornithine or glutamine;    -   X₁₈ is methionine, asparagine or threonine;    -   X₁₉ is tyrosine, 4-methoxy-phenylalanine or 4-amino        phenylalanine;    -   X₂₁ is alanine, glycine or asparagine;    -   X₂₃ is asparagine or glycine;    -   X₂₅ is selected from the group consisting of histidine and        threonine;    -   X₂₉ is selected from the group consisting of alanine, glycine        and serine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid;    -   X₃₃ is selected from the group consisting of aspartic acid and        glutamic acid;    -   X₃₄ is selected from the group consisting of alanine and        threonine;    -   X₄₂ is selected from the group consisting of alanine, lysine,        ornithine and arginine;    -   X₄₅ is tyrosine or phenylalanine and    -   R₅₃ is COOH or CONH₂. In a further embodiment the A chain        comprises the sequence GIVDECCHX₉SCDLX₁₄X₁₅LX₁₇MX₁₉CX₂₁—R₅₃ (SEQ        ID NO: 37), and the B chain comprises the sequence        X₂₅LCGAX₃₀LVDALYLVCGDX₄₂GFX₄₅ (SEQ ID NO: 38) wherein    -   X₉, X₁₄ and X₁₅ are independently ornithine, lysine or arginine;    -   X₁₇ is glutamic acid or glutamine;    -   X₁₉ is tyrosine, 4-methoxy-phenylalanine or 4-amino        phenylalanine;    -   X₂₁ is alanine, glycine or asparagine;    -   X₂₅ is selected from the group consisting of histidine and        threonine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid and glutamic acid;    -   X₄₂ is selected from the group consisting of alanine, lysine,        ornithine and arginine;    -   X₄₅ is tyrosine or phenylalanine and    -   R₅₃ is COOH or CONH₂. In one embodiment the B chain is selected        from the group consisting of HLCGAELVDALYLVCGDX₄₂GFY (SEQ ID NO:        39), GPEHLCGAELVDALYLVCGDX₄₂GFY (SEQ ID NO: 40),        GPEHLCGAELVDALYLVCGDX₄₂GFYFNPKT (SEQ ID NO: 41) and        GPEHLCGAELVDALYLVCGDX₄₂GFYFNKPT (SEQ ID NO: 42), wherein X₄₂ is        selected from the group consisting of ornithine, lysine and        arginine. In a further embodiment the A chain comprises the        sequence GIVDECCHX₉SCDLX₁₄X₁₅LQMYCN-R₅₃ (SEQ ID NO: 43), wherein        X₉, X₁₄ and X₁₅ are independently ornithine, lysine or arginine.

In another embodiment the A chain comprises the sequenceGIVDECCX₈RSCDLYQLENX₁₉CN-R₅₃ (SEQ ID NO: 44) and the B chain comprisesthe sequence R₆₂-X₂₅LCGSHLVDALYLVCGDX₄₂GFX₄₅ (SEQ ID NO: 45)

-   -   wherein    -   X₈ is threonine, histidine or phenylalanine;    -   X₁₉ is tyrosine, 4-methoxy-phenylalanine or 4-amino        phenylalanine;    -   X₂₅ is histidine or threonine;    -   X₄₂ is alanine, ornithine or arginine;    -   X₄₅ is tyrosine histidine, asparagine or phenylalanine;    -   R₆₂ is selected from the group consisting of AYRPSE (SEQ ID NO:        14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide        glycine-proline-glutamic acid, a tripeptide        valine-asparagine-glutamine, a dipeptide proline-glutamic acid,        a dipeptide asparagine-glutamine, glutamine, glutamic acid and a        bond; and R₅₃ is COOH or CONH₂. and    -   R₅₃ is COOH or CONH₂. In a further embodiment X₁₉ is Tyr.

In another embodiment the A chain comprises the sequenceGIVEQCCHSICSLYQLENX₁₉CX₂₁-R₅₃ (SEQ ID NO: 46) orGIVDECCHRSCDLRRLEMX₁₉CX₂₁-R₅₃ (SEQ ID NO: 47); and the B chain comprisesthe sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2), or

GPETLCGAELVDALYLVCGDRGFYFNPKT (SEQ ID NO: 48)

wherein

-   -   X₁₉ is tyrosine, 4-methoxy phenylalanine or        4-amino-phenylalanine; and    -   X₂₁ is alanine, glycine or asparagine.

In another embodiment, the A chain comprises the sequenceGIVEQCCHSICSLYQLENYCX₂₁-R₅₃ (SEQ ID NO: 160) and the B chain comprisesthe sequence FVKQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 147), orFVNQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 148), wherein

X₂₁ is alanine, glycine or asparagine; and

X₂₅ is selected from the group consisting of histidine and threonine;

-   -   X₂₈ is proline, aspartic acid or glutamic acid; and

R₆₃ is selected from the group consisting of YTX₂₈KT (SEQ ID NO: 149),YTKPT (SEQ ID NO: 150), YTX₂₈K (SEQ ID NO: 152), YTKP (SEQ ID NO: 151),YTPK (SEQ ID NO: 70), YTX₂₈, YT, Y and a bond. In one embodiment the Bchain comprises the sequence FVKQX₂₅LCGSHLVEALYLVCGERGFFYTEKT (SEQ IDNO: 162), FVNQX₂₅LCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO: 164),FVNQX₂₅LCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 165) orFVNQX₂₅LCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 161) wherein

X₂₅ is selected from the group consisting of histidine and threonine.

Single Chain Insulin Peptide Agonists

As disclosed herein linking moieties can be used to link human insulin Aand B chains, or analogs or derivatives thereof, wherein the carboxyterminus of the B25 amino acid of the B chain is directly linked to afirst end of a linking moiety, wherein the second end of the linkingmoiety is directly linked to the amino terminus of the A1 amino acid ofthe A chain via the intervening linking moiety.

In accordance with one embodiment the insulin peptide is a single chaininsulin agonist that comprises the general structure B-LM-A wherein Brepresents an insulin B chain, A represents an insulin A chain, and LMrepresents a linking moiety linking the carboxy terminus of the B chainto the amino terminus of the A chain. Suitable linking moieties forjoining the B chain to the A chain are disclosed herein under the headerLinking Moieties for Single Chain-Insulin Analogs and the respectivesubheaders “Peptide linkers”. In one embodiment the linking moietycomprises a linking peptide, and more particularly, in one embodimentthe peptide represents an analog of the IGF-1 C peptide. Additionalexemplary peptide linkers include but are not limited to the sequenceX₅₁X₅₂GSSSX₅₇X₅₈ (SEQ ID NO: 49) or X₅₁X₅₂GSSSX₅₇X₅₈APQT (SEQ ID NO: 50)wherein X₅₁ is selected from the group consisting of glycine, alanine,valine, leucine, isoleucine and proline, X₅₂ is alanine, valine,leucine, isoleucine or proline and X₅₇ or X₅₈ are independentlyarginine, lysine, cysteine, homocysteine, acetyl-phenylalanine orornithine, optionally with a hydrophilic moiety linked to the side chainof the amino acid at position 7 or 8 of the linking moiety (i.e., at theX₅₇ or X₅₈ position). Amino acid positions of the linking moiety aredesignated based on the corresponding position in the native C chain ofIGF 1 (SEQ ID NO: 17). In another embodiment the peptide linking moietycomprises a 29 contiguous amino acid sequence having greater than 70%,80%, 90% sequence identity to SSSSX₅₀APPPSLPSPSRLPGPSDTPILPQX₅₁ (SEQ IDNO: 68), wherein X₅₀ and X₅₁ are independently selected from arginineand lysine. In one embodiment the linking moiety is a non-peptide linkercomprising a relatively short bifunctional non-peptide polymer linkerthat approximates the length of an 8-16 amino acid sequence. In oneembodiment the non-peptide linker has the structure:

wherein m is an integer ranging from 10 to 14 and the linking moiety islinked directly to the B25 amino acid of the B chain. In accordance withone embodiment the non-peptide linking moiety is a polyethylene glycollinker of approximately 4 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 10to 14, 10 to 12 or 11 to 13 monomers.

In one embodiment a FGF21 based insulin conjugate is provided thatcomprises an insulin peptide having the structure: IB-LM-IA, wherein IBcomprises the sequence R₆₂-X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGX₄₁X₄₂GFX₄₅ (SEQ IDNO: 20), LM is a linking moiety as disclosed herein that covalentlylinks IB to IA, and IA comprises the sequenceGIVX₄X₅CCX₈X₉X₁₀CX₁₂LX₁₄X₁₅LX₁₇X₁₈X₁₉CX₂₁—R₅₃(SEQ ID NO: 29), wherein

-   -   X₄ is glutamic acid or aspartic acid;    -   X₅ is glutamine or glutamic acid;    -   X₈ is histidine or phenylalanine;    -   X₉ and X₁₄ are independently selected from arginine, lysine,        ornithine or alanine;    -   X₁₀ is isoleucine or serine;    -   X₁₂ is serine or aspartic acid;    -   X₁₄ is tyrosine, arginine, lysine, ornithine or alanine;    -   X₁₅ is arginine, lysine, ornithine or leucine;    -   X₁₇ is glutamic acid or glutamine;    -   X₁₈ is methionine, asparagine or threonine;    -   X₁₉ is tyrosine, 4-methoxy-phenylalanine or 4-amino        phenylalanine;    -   X₂₁ is alanine, glycine or asparagine;    -   X₂₅ is selected from the group consisting of histidine and        threonine;    -   X₂₉ is selected from the group consisting of alanine, glycine        and serine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid;    -   X₃₃ is selected from the group consisting of aspartic acid and        glutamic acid;    -   X₃₄ is selected from the group consisting of alanine and        threonine;    -   X₄₁ is selected from the group consisting of glutamic acid,        aspartic acid or asparagine;    -   X₄₂ is selected from the group consisting of alanine, lysine,        ornithine and arginine;    -   R₆₂ is selected from the group consisting of AYRPSE (SEQ ID NO:        14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide        glycine-proline-glutamic acid, a tripeptide        valine-asparagine-glutamine, a dipeptide proline-glutamic acid,        a dipeptide asparagine-glutamine, glutamine, glutamic acid and        an N-terminal amine; and    -   R₅₃ is COOH or CONH₂, further wherein the amino acid at the        designation X₄₅ is directly bound to the linking moiety, LM        (i.e., the designation IB-LM-IA as used herein is intended to        represent that the B chain carboxyl terminus and the amino        terminus of the A chain are directly linked to the linking        moiety LM without any further intervening amino acids).

In one embodiment the linking moiety (LM) comprises an amino acidsequence of no more than 17 amino acids in length. In one embodiment thelinking moiety comprises the sequence X₅₁X₅₂GSSSX₅₇X₅₈ (SEQ ID NO: 49)or X₅₁X₅₂GSSSX₅₇X₅₈APQT (SEQ ID NO: 50) wherein X₅₁ is selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine andproline, X₅₂ is alanine, valine, leucine, isoleucine or proline and X₅₇or X₅₈ are independently arginine, lysine, cysteine, homocysteine,acetyl-phenylalanine or ornithine, optionally with a hydrophilic moietylinked to the side chain of the amino acid at position 7 or 8 of thelinking moiety (i.e., at the X₅₇ or X₅₈ position). Amino acid positionsof the linking moiety are designated based on the corresponding positionin the native C chain of IGF 1 (SEQ ID NO: 17). In one embodiment LM isGAGSSSRRAPQT (SEQ ID NO: 23) or GAGSSSRR (SEQ ID NO: 22).

In another embodiment the linking moiety comprises a 29 contiguous aminoacid sequence, directly linked to the carboxy terminal amino acid of theB chain, wherein said 29 contiguous amino acid sequence has greater than70%, 80%, 90% sequence identity to SSSSX₅₀APPPSLPSPSRLPGPSDTPILPQX₅₁(SEQ ID NO: 68), wherein X₅₀ and X₅₁ are independently selected fromarginine and lysine. In one embodiment the linking peptide comprises atotal of 29 to 158 or 29 to 58 amino acids and comprises the sequence ofSEQ ID NO: 68. In another embodiment the linking moiety comprises a 29contiguous amino acid sequence, directly linked to the carboxy terminalamino acid of the B chain, wherein said 29 contiguous amino acidsequence has greater than 90% sequence identity toSSSSX₅₀APPPSLPSPSRLPGPSDTPILPQX₅₁ (SEQ ID NO: 68), wherein X₅₀ and X₅₁are independently selected from arginine and lysine. In one embodimentthe linking moiety comprises the sequence SSSSRAPPPSLPSPSRLPGPSDTPILPQK(SEQ ID NO: 51) or SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 52)optionally with one or two amino acid substitutions.

In accordance with one embodiment a single chain insulin agonistpolypeptide is provided comprising a B chain and A chain of humaninsulin, or analogs or derivative thereof, wherein the last five carboxyamino acids of the native B chain are deleted (i.e., B26-B30), and aminoacid B25 is linked to amino acid A1 of the A chain via an interveninglinking moiety. In one embodiment the linking moiety comprises thestructure:

wherein m is an integer ranging from 10 to 14 and the linking moiety islinked directly to the B25 amino acid of the B chain.

In one embodiment an FGF21 based insulin conjugate is providedcomprising an insulin peptide having the general formula IB-LM-IAwherein IB comprises the sequence GPEHLCGAX₃₀LVDALYLVCGDX₄₂GFYFNX₄₈X₄₉(SEQ ID NO: 163);

LM comprises the sequence SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51),SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 52), GYGSSSRR (SEQ ID NO: 18),GAGSSSRRAPQT (SEQ ID NO: 23) or GAGSSSRR (SEQ ID NO: 22); and

IA comprises the sequence GIVDECCX₈X₉SCDLX₁₄X₁₅LX₁₇X₁₈X₁₉CX₂₁—R₅₃ (SEQID NO: 35) wherein

-   -   X₈ is histidine or phenylalanine;    -   X₉ is arginine, ornithine or alanine;    -   X₁₄ and X₁₅ are both arginine;    -   X₁₇ is glutamic acid;    -   X₁₉ is tyrosine, 4-methoxy-phenylalanine or 4-amino        phenylalanine;    -   X₂₁ is alanine or asparagine;    -   X₂₅ is histidine or threonine;    -   X₃₀ is selected from the group consisting of histidine, aspartic        acid, glutamic acid, homocysteic acid and cysteic acid;    -   X₄₂ is selected from the group consisting of alanine, ornithine        and arginine;    -   R₅₃ is COOH.

Linking Moieties for Single Chain Insulin Analogs

Peptide Linkers

In accordance with one embodiment the linking moiety is a peptide orpeptidomimetic of 6-18, 8-18, 8-17, 8-12, 8-10, 13-17 or 13-15 aminoacids (or amino acid analogs or derivatives thereof). In one embodimentthe linking moiety is 8 to 17 amino acids in length and comprises thesequence X₅₁X₅₂GSSSRR (SEQ ID NO: 53) wherein X₅₁ is selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline and methionine, and X₅₂ is a non-aromatic amino acid, includingfor example, alanine. In one embodiment the linking moiety is 8 to 17amino acids in length and comprises a sequence that differs fromX₅₁X₅₂GSSSRR (SEQ ID NO: 53) by a single amino acid substitution whereinthe amino acid substitution is an amino acid that is pegylated at itsside chain, further wherein X₅₁ is selected from the group consisting ofglycine, alanine, valine, leucine, isoleucine, proline and methionine,and X₅₂ is a non-aromatic amino acid, including for example, alanine.

In accordance with one embodiment the linking moiety is a derivative ofthe IGF 1 C chain sequence (GYGSSSRRAPQT; SEQ ID NO: 17). In oneembodiment the derivative is a peptide that differs from SEQ ID NO: 17by a single amino acid substitution of a lysine, cysteine ornithine,homocysteine, or acetyl-phenylalanine residue, and in a furtherembodiment the lysine, cysteine ornithine, homocysteine, oracetyl-phenylalanine amino acid is pegylated. In one further embodimentthe linking moiety is a peptide that differs from SEQ ID NO: 17 by asingle lysine substitution. In one specific embodiment the substitutionis made at position 8 of SEQ ID NO: 17. Applicants have discovered thatuse of the IGF 1 C chain sequence and analogs thereof as a linkingmoiety will generate a single chain insulin polypeptide that has nearwild type insulin activity. Furthermore, use of a IGF 1 C chain sequenceanalog as the linking moiety, wherein position 2 of the IGF 1 C chainsequence is modified, or the carboxy terminal four amino acids aredeleted from the IGF 1 C chain sequence, produces a single chain insulinpolypeptide that is selective for insulin (i.e., has a higher bindingand/or activity at the insulin receptor compared to the IGF-1 receptor).In one embodiment the single chain insulin polypeptide has 5×, 10×, 20×,30×, 40×, or 50× higher affinity or activity at the insulin receptorrelative to the IGF-1 receptor.

In accordance with one embodiment the linking moiety is a derivative ofthe IGF 1 C chain sequence (GYGSSSRRAPQT; SEQ ID NO: 17) and comprises anon-native sequence that differs from GYGSSSRR (SEQ ID NO: 18) orGAGSSSRRAPQT (SEQ ID NO: 23) by 1 to 3 amino acid substitutions, or 1 to2 amino acid substitutions. In one embodiment at least one of the aminoacid substitutions is a lysine or cysteine substitution, and in oneembodiment the amino acid substitutions are conservative amino acidsubstitutions. In one embodiment the linking moiety is a peptide (orpeptidomimetic) of 8 to 17 amino acids comprising a non-native aminoacid sequence that differs from GYGSSSRR (SEQ ID NO: 18) or GAGSSSRRAPQT(SEQ ID NO: 23) by 1 amino acid substitution, including for examplesubstitution with a lysine or cysteine. In one embodiment the linkingmoiety comprises the sequence GYGSSSRR (SEQ ID NO: 18) or GAGSSSRRAPQT(SEQ ID NO: 23). In one embodiment the linking moiety comprises thesequence GAGSSSRX₅₈APQT (SEQ ID NO: 54), GYGSSSX₅₇X₅₈APQT (SEQ ID NO:69), or an amino acid that differs from SEQ ID NO: 54 by a single aminoacid substitution, wherein X₅₇ is arginine and X₅₈ is arginine,ornithine or lysine, and in a further embodiment a polyethylene glycolchain is linked to the side chain of the amino acid at position 8 ofsaid linking moiety. In another embodiment the linking moiety comprisesthe sequence GX₅₂GSSSRX₅₈APQT (SEQ ID NO: 55), wherein X₅₂ is anynon-aromatic amino acid, including for example, alanine, valine,leucine, isoleucine or proline, and X₅₈ represents an amino acid thathas a polyethylene chain covalently linked to its side chain. In oneembodiment X₅₈ is a pegylated lysine.

In another embodiment, the linking moiety is an 8 to 17 amino acidsequence comprising the sequence GX₅₂GSSSRR (SEQ ID NO: 56), wherein X₅₂is any amino acid, a peptidomimetic of SEQ ID NO: 31, or an analogthereof that differs from SEQ ID NO: 31 by a single amino acidsubstitution at any of positions 1, 3, 4, 5, 6, 7 or 8 of SEQ ID NO: 31,with the proviso that when the linking peptide is longer than 8 aminoacids X₅₂ is other than tyrosine. In accordance with one embodiment thelinking moiety comprises an 8-17 amino acid sequence selected from thegroup consisting of GYGSSSRR (SEQ ID NO: 18), GAGSSSRR (SEQ ID NO: 22),GAGSSSRRA (SEQ ID NO: 57), GAGSSSRRAP (SEQ ID NO: 58), GAGSSSRRAPQ (SEQID NO: 59), GAGSSSRRAPQT (SEQ ID NO: 23), PYGSSSRR (SEQ ID NO: 61),PAGSSSRR (SEQ ID NO: 62), PAGSSSRRA (SEQ ID NO: 63), PAGSSSRRAP (SEQ IDNO: 64), PAGSSSRRAPQ (SEQ ID NO: 65), PAGSSSRRAPQT (SEQ ID NO: 66). Inaccordance with one embodiment the linking moiety comprises an aminoacid sequence that differs from GYGSSSRR (SEQ ID NO: 18), GAGSSSRR (SEQID NO: 22), GAGSSSRRA (SEQ ID NO: 57), GAGSSSRRAP (SEQ ID NO: 58),GAGSSSRRAPQ (SEQ ID NO: 59), GAGSSSRRAPQT (SEQ ID NO: 23), PYGSSSRR (SEQID NO: 61), PAGSSSRR (SEQ ID NO: 62), PAGSSSRRA (SEQ ID NO: 63),PAGSSSRRAP (SEQ ID NO: 64), PAGSSSRRAPQ (SEQ ID NO: 65), PAGSSSRRAPQT(SEQ ID NO: 66) by a single pegylated amino acid including for example apegylated lysine or pegylated cysteine amino acid substitution. In oneembodiment the pegylated amino acid is at position 8 of the linkingmoiety.

In one embodiment a peptide sequence named C-terminal peptide (CTP:SSSSKAPPPSLPSPSRLPGPSDTPILPQR; SEQ ID NO: 52), which is prone toO-linked hyperglycosylation when the protein is expressed in aeukaryotic cellular expression system, can be used as a linker peptide.Surprisingly, applicants have discovered that the CTP peptide can beused to connect the B and A chains of insulin to form a single chaininsulin analog while still maintaining high in vitro potency in a mannerthat the native proinsulin C-peptide cannot. In one embodiment a FGF21based insulin conjugate is prepared comprising an insulin peptide havingthe carboxy terminus of the B chain linked to the amino terminus of theA chain via a CTP peptide. In another embodiment an insulin analog isprovided as a two-chain construct with the CTP covalently linked to theC-terminus of the B-chain and/or the amino terminus of the B chain. Invitro and in vivo characterization reveals the CTP modified insulinanalogs to have high potency in the absence of glycosylation, thusproviding a mechanism to extend insulin action that is based onglycosylation, a natural approach to longer duration proteins.

Applicants have discovered that the primary sequence of the CTP peptidedoes not appear to be critical. Accordingly, in one embodiment thelinking moiety comprises a peptide having a length of at least 18 aminoacids that shares a similar amino acid content. In one embodiment thelinking moiety comprises an analog of (SEQ ID NO: 68), wherein saidanalog differs from (SEQ ID NO: 68) by 1, 2, 3, 4, 5 or 6 amino acidsubstitutions. In one embodiment the linking peptide comprises a CTPpeptide wherein amino acid substitutions are made at one or morepositions selected from positions 1, 2, 3, 4, 10, 13, 15, and 21 of (SEQID NO: 68). In one embodiment the linking moiety comprises a 29contiguous amino acid sequence, directly linked to the carboxy terminalamino acid of the B chain, wherein said 29 contiguous amino acidsequence has greater than 60, 80 or 90% sequence identity toSSSSX₅₀APPPSLPSPSRLPGPSDTPILPQX_(5i) (SEQ ID NO: 68), with the provisothat the sequence does not comprise a 15 amino acid sequence identicalto a 15 amino acid sequence contained within SEQ ID NO 53. In anotherembodiment the linking moiety comprises a 29 contiguous amino acidsequence, directly linked to the carboxy terminal amino acid of the Bchain, wherein at least 58% of the amino acids comprising the 29contiguous amino acid sequence are selected from the group consisting ofserine and proline.

In another embodiment the linking moiety comprises a 29 contiguous aminoacid sequence, directly linked to the carboxy terminal amino acid of theB chain, wherein said 29 contiguous amino acid sequence has greater than70%, 80%, 90% sequence identity to SSSSX₅₀APPPSLPSPSRLPGPSDTPILPQX_(5i)(SEQ ID NO: 68), wherein X₅₀ and X₅₁ are independently selected fromarginine and lysine, with the proviso that the sequence does notcomprise a 15 amino acid sequence identical to a 15 amino acid sequencecontained within SEQ ID NO 53. In another embodiment the linking moietycomprises a 29 contiguous amino acid sequence, directly linked to thecarboxy terminal amino acid of the B chain, wherein said 29 contiguousamino acid sequence is an analog of (SEQ ID NO: 52), wherein said analogdiffers from (SEQ ID NO: 52) only by 1, 2, 3, 4, 5 or 6 amino acidmodification, and in a further embodiment the amino acid modificationsare conservative amino acid substitutions. In another embodiment thelinking moiety comprises a 29 contiguous amino acid sequence, directlylinked to the carboxy terminal amino acid of the B chain, wherein said29 contiguous amino acid sequence is an analog of (SEQ ID NO: 52),wherein said analog differs from (SEQ ID NO: 52) only by 1, 2 or 3 aminoacid substitutions.

Applicants have also found that multiple copies of the CTP peptide canbe used as the linking peptide in single chain analogs and/or linked tothe amino terminus of the B chain in single chain or two chain insulinanalogs. The multiple copies of the CTP peptide can be identical or candiffer in sequence and can be arranged in a head to tail or head to headorientation. In accordance with one embodiment an insulin analog isprovided comprising a CTP peptide having the sequence(SSSSX₅₀APPPSLPSPSRLPGPSDTPILPQX₅₁)_(n)(SEQ ID NO: 68), wherein n is aninteger selected from the group consisting of 1, 2, 3 and 4 and X₅₀ andX₅₁ are independently selected from arginine and lysine.

In one embodiment the CTP peptide comprises the sequenceSSSSX₅₀APPPSLPSPSRLPGPSDTPILPQX₅₁ (SEQ ID NO: 68), wherein X₅₀ and X₅₁are independently selected from arginine and lysine. In anotherembodiment the CTP peptide comprises a sequence selected from the groupconsisting of SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51),SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 52) orSSSSRAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 67), and in a furtherembodiment the CTP peptide comprises the sequenceSSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51).

Structure of L

In some embodiments, L is a bond. In these embodiments, Q and Y areconjugated together by reacting a nucleophilic reactive moiety on Q withand electrophilic reactive moiety on Y. In alternative embodiments, Qand Y are conjugated together by reacting an electrophilic reactivemoiety on Q with a nucleophilic moiety on Y. In exemplary embodiments, Lis an amide bond that forms upon reaction of an amine on Q (e.g. anε-amine of a lysine residue) with a carboxyl group on Y. In alternativeembodiments, Q and or Y are derivatized with a derivatizing agent beforeconjugation.

In some embodiments, L is a linking group. In some embodiments, L is abifunctional linker and comprises only two reactive groups beforeconjugation to Q and Y. In embodiments where both Q and Y haveelectrophilic reactive groups, L comprises two of the same or twodifferent nucleophilic groups (e.g. amine, hydroxyl, thiol) beforeconjugation to Q and Y. In embodiments where both Q and Y havenucleophilic reactive groups, L comprises two of the same or twodifferent electrophilic groups (e.g. carboxyl group, activated form of acarboxyl group, compound with a leaving group) before conjugation to Qand Y. In embodiments where one of Q or Y has a nucleophilic reactivegroup and the other of Q or Y has an electrophilic reactive group, Lcomprises one nucleophilic reactive group and one electrophilic groupbefore conjugation to Q and Y.

L can be any molecule with at least two reactive groups (beforeconjugation to Q and Y) capable of reacting with each of Q and Y. Insome embodiments L has only two reactive groups and is bifunctional. L(before conjugation to the peptides) can be represented by Formula VI:

wherein W and J are independently nucleophilic or electrophilic reactivegroups. In some embodiments W and J are either both nucleophilic groupsor both electrophilic groups. In some embodiments one of W or J is anucleophilic group and the other of W or J is an electrophilic group.

In some embodiments, L comprises a chain of atoms from 1 to about 60, or1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or10 to 20 atoms long. In some embodiments, the chain atoms are all carbonatoms. In some embodiments, the chain atoms in the backbone of thelinker are selected from the group consisting of C, O, N, and S. Chainatoms and linkers may be selected according to their expected solubility(hydrophilicity) so as to provide a more soluble conjugate. In someembodiments, L provides a functional group that is subject to cleavageby an enzyme or other catalyst or hydrolytic conditions found in thetarget tissue or organ or cell. In some embodiments, the length of L islong enough to reduce the potential for steric hindrance.

In some embodiments, the linking group is hydrophilic such as, forexample, polyalkylene glycol. Before conjugation to the peptides of thecomposition, the hydrophilic linking group comprises at least tworeactive groups (W and J), as described herein and as shown below:

In specific embodiments, the linking group is polyethylene glycol (PEG).The PEG in certain embodiments has a molecular weight of about 100Daltons to about 10,000 Daltons, e.g. about 500 Daltons to about 5000Daltons. The PEG in some embodiments has a molecular weight of about10,000 Daltons to about 40,000 Daltons.

In some embodiments, the hydrophilic linking group comprises either amaleimido or an iodoacetyl group and either a carboxylic acid or anactivated carboxylic acid (e.g. NHS ester) as the reactive groups. Inthese embodiments, the maleimido or iodoacetyl group can be coupled to athiol moiety on Q or Y and the carboxylic acid or activated carboxylicacid can be coupled to an amine on Q or Y with or without the use of acoupling reagent. Any appropriate coupling agent known to one skilled inthe art can be used to couple the carboxylic acid with the amine. Insome embodiments, the linking group is maleimido-PEG(20 kDa)-COOH,iodoacetyl-PEG(20 kDa)-COOH, maleimido-PEG(20 kDa)-NHS, oriodoacetyl-PEG(20 kDa)-NHS.

In some embodiments, the linking group is comprised of an amino acid, adipeptide, a tripeptide, or a polypeptide, wherein the amino acid,dipeptide, tripeptide, or polypeptide comprises at least two activatinggroups, as described herein. In some embodiments, the linking group (L)comprises a moiety selected from the group consisting of: amino, ether,thioether, maleimido, disulfide, amide, ester, thioester, alkene,cycloalkene, alkyne, trizoyl, carbamate, carbonate, cathepsinB-cleavable, and hydrazone. In some embodiments, the linking group is anamino acid selected from the group Asp, Glu, homoglutamic acid,homocysteic acid, cysteic acid, gamma-glutamic acid. In someembodiments, the linking group is a dipeptide selected from the groupconsisting of: Ala-Ala, β-Ala-β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyricacid-γ-aminobutyric acid, and γ-Glu-γ-Glu. In one embodiment L comprisesgamma-glutamic acid.

In embodiments where Q and Y are conjugated together by reacting acarboxylic acid with an amine, an activating agent can be used to forman activated ester of the carboxylic acid. The activated ester of thecarboxylic acid can be, for example, N-hydroxysuccinimide (NHS),tosylate (Tos), mesylate, triflate, a carbodiimide, or ahexafluorophosphate. In some embodiments, the carbodiimide is1,3-dicyclohexylcarbodiimide (DCC), 1,1′-carbonyldiimidazole (CDI),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or1,3-diisopropylcarbodiimide (DICD). In some embodiments, thehexafluorophosphate is selected from a group consisting ofhexafluorophosphate benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU), ando-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU).

In some embodiments, Q comprises a nucleophilic reactive group (e.g. theamino group, thiol group, or hydroxyl group of the side chain of lysine,cysteine or serine) that is capable of conjugating to an electrophilicreactive group on Y or L. In some embodiments, Q comprises anelectrophilic reactive group (e.g. the carboxylate group of the sidechain of Asp or Glu) that is capable of conjugating to a nucleophilicreactive group on Y or L. In some embodiments, Q is chemically modifiedto comprise a reactive group that is capable of conjugating directly toY or to L. In some embodiments, Q is modified at the C-terminal tocomprise a natural or nonnatural amino acid with a nucleophilic sidechain, such as an amino acid represented by Formula I, Formula II, orFormula III, as previously described herein (see Acylation andalkylation). In exemplary embodiments, the C-terminal amino acid of Q isselected from the group consisting of lysine, ornithine, serine,cysteine, and homocysteine. For example, the C-terminal amino acid of Qcan be modified to comprise a lysine residue. In some embodiments, Q ismodified at the C-terminal amino acid to comprise a natural ornonnatural amino acid with an electrophilic side chain such as, forexample, Asp and Glu. In some embodiments, an internal amino acid of Qis substituted with a natural or nonnatural amino acid having anucleophilic side chain, such as an amino acid represented by Formula I,Formula II, or Formula III, as previously described herein (seeAcylation and alkylation). In exemplary embodiments, the internal aminoacid of Q that is substituted is selected from the group consisting oflysine, ornithine, serine, cysteine, and homocysteine. For example, aninternal amino acid of Q can be substituted with a lysine residue. Insome embodiments, an internal amino acid of Q is substituted with anatural or nonnatural amino acid with an electrophilic side chain, suchas, for example, Asp and Glu.

In some embodiments, Y comprises a reactive group that is capable ofconjugating directly to Q or to L. In some embodiments, Y comprises anucleophilic reactive group (e.g. amine, thiol, hydroxyl) that iscapable of conjugating to an electrophilic reactive group on Q or L. Insome embodiments, Y comprises electrophilic reactive group (e.g.carboxyl group, activated form of a carboxyl group, compound with aleaving group) that is capable of conjugating to a nucleophilic reactivegroup on Q or L.

Stability of L In Vivo

In some embodiments, L is stable in vivo. In some embodiments, L isstable in blood serum for at least 5 minutes, e.g. less than 25%, 20%,15%, 10% or 5% of the conjugate is cleaved when incubated in serum for aperiod of 5 minutes. In other embodiments, L is stable in blood serumfor at least 10, or 20, or 25, or 30, or 60, or 90, or 120 minutes, or3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 or 24 hours. In these embodiments, Ldoes not comprise a functional group that is capable of undergoinghydrolysis in vivo. In some exemplary embodiments, L is stable in bloodserum for at least about 72 hours. Nonlimiting examples of functionalgroups that are not capable of undergoing significant hydrolysis in vivoinclude amides, ethers, and thioethers. For example, the followingcompound is not capable of undergoing significant hydrolysis in vivo:

In some embodiments, L is hydrolyzable in vivo. In these embodiments, Lcomprises a functional group that is capable of undergoing hydrolysis invivo. Nonlimiting examples of functional groups that are capable ofundergoing hydrolysis in vivo include esters, anhydrides, andthioesters. For example the following compound is capable of undergoinghydrolysis in vivo because it comprises an ester group:

In some exemplary embodiments L is labile and undergoes substantialhydrolysis within 3 hours in blood plasma at 37° C., with completehydrolysis within 6 hours. In some exemplary embodiments, L is notlabile.

In some embodiments, L is metastable in vivo. In these embodiments, Lcomprises a functional group that is capable of being chemically orenzymatically cleaved in vivo (e.g., an acid-labile, reduction-labile,or enzyme-labile functional group), optionally over a period of time. Inthese embodiments, L can comprise, for example, a hydrazone moiety, adisulfide moiety, or a cathepsin-cleavable moiety. When L is metastable,and without intending to be bound by any particular theory, the Q-L-Yconjugate is stable in an extracellular environment, e.g., stable inblood serum for the time periods described above, but labile in theintracellular environment or conditions that mimic the intracellularenvironment, so that it cleaves upon entry into a cell. In someembodiments when L is metastable, L is stable in blood serum for atleast about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or48 hours, for example, at least about 48, 54, 60, 66, or 72 hours, orabout 24-48, 48-72, 24-60, 36-48, 36-72, or 48-72 hours.

Pegylation of Insulin Peptides

Applicants have discovered that covalent linkage of a hydrophilic moietyto the insulin analogs disclosed herein provide analogs having sloweronset, extended duration and exhibit a basal profile of activity. In oneembodiment, the insulin peptides disclosed herein are further modifiedto comprise a hydrophilic moiety covalently linked to the side chain ofan amino acid at a position selected from the group consisting of A9,A14 and A15 of the A chain or at the N-terminal alpha amine of the Bchain (e.g. at position B1 for insulin based B chain or position B2 forIGF-1 based B chain) or at the side chain of an amino acid at positionB1, B2, B10, B22, B28 or B29 of the B chain or at any position of thelinking moiety that links the A chain and B chain. In exemplaryembodiments, this hydrophilic moiety is covalently linked to a Lys, Cys,Orn, homocysteine, or acetyl-phenylalanine residue at any of thesepositions. In one embodiment the hydrophilic moiety is covalently linkedto the side chain of an amino acid of the linking moiety.

Exemplary hydrophilic moieties include polyethylene glycol (PEG), forexample, of a molecular weight of about 1,000 Daltons to about 40,000Daltons, or about 20,000 Daltons to about 40,000 Daltons. Additionalsuitable hydrophilic moieties include, polypropylene glycol,polyoxyethylated polyols (e.g., POG), polyoxyethylated sorbitol,polyoxyethylated glucose, polyoxyethylated glycerol (POG),polyoxyalkylenes, polyethylene glycol propionaldehyde, copolymers ofethylene glycol/propylene glycol, monomethoxy-polyethylene glycol,mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol,carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, poly (beta-amino acids) (either homopolymers orrandom copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers (PPG) and other polyakylene oxides,polypropylene oxide/ethylene oxide copolymers, colonic acids or otherpolysaccharide polymers, Ficoll or dextran and mixtures thereof.

Hydrophilic moieties such as polyethylene glycol can be attached to theFGF21 based conjugates of the present disclosure under any suitableconditions used to react a protein with an activated polymer molecule.Any means known in the art can be used, including via acylation,reductive alkylation, Michael addition, thiol alkylation or otherchemoselective conjugation/ligation methods through a reactive group onthe PEG moiety (e.g., an aldehyde, amino, ester, thiol, α-haloacetyl,maleimido or hydrazino group) to a reactive group on the target compound(e.g., an aldehyde, amino, ester, thiol, α-haloacetyl, maleimido orhydrazino group). Activating groups which can be used to link the watersoluble polymer to one or more proteins include without limitationsulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine,oxirane and 5-pyridyl. If attached to the peptide by reductivealkylation, the polymer selected should have a single reactive aldehydeso that the degree of polymerization is controlled. See, for example,Kinstler et al., Adv. Drug. Delivery Rev. 54: 477-485 (2002); Roberts etal., Adv. Drug Delivery Rev. 54: 459-476 (2002); and Zalipsky et al.,Adv. Drug Delivery Rev. 16: 157-182 (1995).

Acylation

In some embodiments, the FGF21 based conjugate is modified to comprisean acyl group. The acyl group can be covalently linked directly to anamino acid of the bioactive component of the conjugate (ie., the NHRligand or the insulin component of conjugate), or indirectly to an aminoacid of the NHR ligand or insulin peptide via a spacer, wherein thespacer is positioned between the amino acid of the bioactive componentof the conjugate and the acyl group. The conjugate may be acylated atthe same amino acid position where a hydrophilic moiety is linked, or ata different amino acid position. For example, acylation may occur at anyposition including any of amino acid of the conjugate, provided that theactivity exhibited by the non-acylated conjugate is retained uponacylation.

In one specific aspect of the invention, an FGF21 based insulinconjugate is modified to comprise an acyl group by direct acylation ofan amine, hydroxyl, or thiol of a side chain of an amino acid of theFGF21 based insulin conjugate. In some embodiments, the conjugate isdirectly acylated through the side chain amine, hydroxyl, or thiol of anamino acid. In some embodiments, acylation is at position B28 or B29 ofthe insulin moiety of the conjugate (according to the amino acidnumbering of the native insulin A and B chain sequences). In thisregard, an insulin analog can be provided that has been modified by oneor more amino acid substitutions in the A or B chain sequence, includingfor example at positions A14, A15, B1, B2, B10, B22, B28 or B29(according to the amino acid numbering of the native insulin A and Bchain sequences) or at any position of the linking moiety with an aminoacid comprising a side chain amine, hydroxyl, or thiol. In some specificembodiments of the invention, the direct acylation of the insulinpeptide occurs through the side chain amine, hydroxyl, or thiol of theamino acid at position B28 or B29 (according to the amino acid numberingof the native insulin A and B chain sequences).

In accordance with one embodiment, the acylated conjugates comprise aspacer between the peptide and the acyl group. In some embodiments, theFGF21 based conjugate is covalently bound to the spacer, which iscovalently bound to the acyl group. In some exemplary embodiments, theconjugate is modified to comprise an acyl group by acylation of anamine, hydroxyl, or thiol of a spacer, which spacer is attached to aside chain of an amino acid of the conjugate. The amino acid of theFGF21 based conjugate to which the spacer is attached can be any aminoacid comprising a moiety which permits linkage to the spacer. Forexample, an amino acid comprising a side chain —NH₂, —OH, or —COOH(e.g., Lys, Orn, Ser, Asp, or Glu) is suitable.

In some embodiments, the spacer between the FGF21 based conjugate andthe acyl group is an amino acid comprising a side chain amine, hydroxyl,or thiol (or a dipeptide or tripeptide comprising an amino acidcomprising a side chain amine, hydroxyl, or thiol). In some embodiments,the spacer comprises a hydrophilic bifunctional spacer. In a specificembodiment, the spacer comprises an amino poly(alkyloxy)carboxylate. Inthis regard, the spacer can comprise, for example,NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1 to 6 andn is any integer from 2 to 12, such as, e.g., 8-amino-3,6-dioxaoctanoicacid, which is commercially available from Peptides International, Inc.(Louisville, Ky.). In one embodiment, the hydrophilic bifunctionalspacer comprises two or more reactive groups, e.g., an amine, ahydroxyl, a thiol, and a carboxyl group or any combinations thereof. Incertain embodiments, the hydrophilic bifunctional spacer comprises ahydroxyl group and a carboxylate. In other embodiments, the hydrophilicbifunctional spacer comprises an amine group and a carboxylate. In otherembodiments, the hydrophilic bifunctional spacer comprises a thiol groupand a carboxylate.

In some embodiments, the spacer between peptide the FGF21 basedconjugate and the acyl group is a hydrophobic bifunctional spacer.Hydrophobic bifunctional spacers are known in the art. See, e.g.,Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego,Calif., 1996), which is incorporated by reference in its entirety. Inaccordance with certain embodiments the bifunctional spacer can be asynthetic or naturally occurring amino acid comprising an amino acidbackbone that is 3 to 10 atoms in length (e.g., 6-amino hexanoic acid,5-aminovaleric acid, 7-aminoheptanoic acid, and 8-aminooctanoic acid).Alternatively, the spacer can be a dipeptide or tripeptide spacer havinga peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) inlength. Each amino acid of the dipeptide or tripeptide spacer attachedto the FGF21 based insulin conjugate can be independently selected fromthe group consisting of: naturally-occurring and/or non-naturallyoccurring amino acids, including, for example, any of the D or L isomersof the naturally-occurring amino acids (Ala, Cys, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr), or anyD or L isomers of the non-naturally occurring amino acids selected fromthe group consisting of: β-alanine (β-Ala), N-α-methyl-alanine (Me-Ala),aminobutyric acid (Abu), α-aminobutyric acid (γ-Abu), aminohexanoic acid(ε-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid,aminopiperidinecarboxylic acid, aminoserine (Ams),aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, β-aspartic acid (β-Asp), azetidine carboxylic acid,3-(2-benzothiazolyl)alanine, α-tert-butylglycine,2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine(Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab),diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),dimethylthiazolidine (DMTA), γ-Glutamic acid (γ-Glu), homoserine (Hse),hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide,methyl-isoleucine (MeIle), isonipecotic acid (Isn), methyl-leucine(MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone(Met(O2)), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline(Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine(Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO2)),4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg),piperidinylalanine, piperidinylglycine, 3,4-dehydroproline,pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec),U-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA),1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic),tetrahydropyranglycine, thienylalanine (Thi), U-Benzyl-phosphotyrosine,O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine,O-(bis-dimethylamino-phosphono)-tyrosine, tyrosine sulfatetetrabutylamine, methyl-valine (MeVal), 1-amino-1-cyclohexane carboxylicacid (Acx), aminovaleric acid, beta-cyclopropyl-alanine (Cpa),propargylglycine (Prg), allylglycine (Alg),2-amino-2-cyclohexyl-propanoic acid (2-Cha), tertbutylglycine (Tbg),vinylglycine (Vg), 1-amino-1-cyclopropane carboxylic acid (Acp),1-amino-1-cyclopentane carboxylic acid (Acpe), alkylated3-mercaptopropionic acid, 1-amino-1-cyclobutane carboxylic acid (Acb).In some embodiments the dipeptide spacer is selected from the groupconsisting of: Ala-Ala, β-Ala-β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyricacid-γ-aminobutyric acid, and γ-Glu-γ-Glu.

The FGF21 based conjugate can be modified to comprise an acyl group byacylation of a long chain alkane of any size and can comprise any lengthof carbon chain. The long chain alkane can be linear or branched. Incertain aspects, the long chain alkane is a C₄ to C₃₀ alkane. Forexample, the long chain alkane can be any of a C₄ alkane, C₆ alkane, C₈alkane, C₁₀ alkane, C₁₂ alkane, C₁₄ alkane, C₁₆ alkane, C₁₈ alkane, C₂₀alkane, C₂₂ alkane, C₂₄ alkane, C₂₆ alkane, C₂₈ alkane, or a C₃₀ alkane.In some embodiments, the long chain alkane comprises a C₈ to C₂₀ alkane,e.g., a C₁₄ alkane, C₁₆ alkane, or a C₁₈ alkane.

In some embodiments, an amine, hydroxyl, or thiol group of the FGF21based conjugate is acylated with a cholesterol acid. In a specificembodiment, the peptide is linked to the cholesterol acid through analkylated des-amino Cys spacer, i.e., an alkylated 3-mercaptopropionicacid spacer. Suitable methods of peptide acylation via amines,hydroxyls, and thiols are known in the art. See, for example, Miller,Biochem Biophys Res Commun 218: 377-382 (1996); Shimohigashi andStammer, Int J Pept Protein Res 19: 54-62 (1982); and Previero et al.,Biochim Biophys Acta 263: 7-13 (1972) (for methods of acylating througha hydroxyl); and San and Silvius, J Pept Res 66: 169-180 (2005) (formethods of acylating through a thiol); Bioconjugate Chem. “ChemicalModifications of Proteins: History and Applications” pages 1, 2-12(1990); Hashimoto et al., Pharmacuetical Res. “Synthesis of PalmitoylDerivatives of Insulin and their Biological Activity” Vol. 6, No: 2 pp.171-1′76 (1989).

The acyl group of the acylated peptide the FGF21 based conjugate can beof any size, e.g., any length carbon chain, and can be linear orbranched. In some specific embodiments of the invention, the acyl groupis a C₄ to C₃₀ fatty acid. For example, the acyl group can be any of aC₄ fatty acid, C₆ fatty acid, C₈ fatty acid, C₁₀ fatty acid, C₁₂ fattyacid, C₁₄ fatty acid, C₁₆ fatty acid, C₁₈ fatty acid, C₂₀ fatty acid,C₂₂ fatty acid, C₂₄ fatty acid, C₂₆ fatty acid, C₂₈ fatty acid, or a C₃₀fatty acid. In some embodiments, the acyl group is a C₈ to C₂₀ fattyacid, e.g., a C₁₄ fatty acid or a C₁₆ fatty acid.

In an alternative embodiment, the acyl group is a bile acid. The bileacid can be any suitable bile acid, including, but not limited to,cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid,taurocholic acid, glycocholic acid, and cholesterol acid.

Alkylation

In some embodiments, the FGF21 based conjugate is modified to comprisean alkyl group. The alkyl group can be covalently linked directly to anamino acid of the conjugate analog, or indirectly to an amino acid ofthe FGF21 based conjugate via a spacer, wherein the spacer is positionedbetween the amino acid of the FGF21 based conjugate and the alkyl group.The alkyl group can be attached to the FGF21 based conjugate via anether, thioether, or amino linkage. For example, the FGF21 basedconjugate may be alkylated at the same amino acid position where ahydrophilic moiety is linked, or at a different amino acid position.

Alkylation can be carried out at any position within the FGF21 basedconjugate, including for example in the C-terminal region of the B chainor at a position in the linking moiety, provided that FGF activity isretained. In a specific aspect of the invention, the FGF21 basedconjugate is modified to comprise an alkyl group by direct alkylation ofan amine, hydroxyl, or thiol of a side chain of an amino acid of theFGF21 based conjugate. In some embodiments, the FGF21 based conjugate isdirectly alkylated through the side chain amine, hydroxyl, or thiol ofan amino acid. In some specific embodiments of the invention, the directalkylation of an FGF21 based insulin conjugate occurs through the sidechain amine, hydroxyl, or thiol of the amino acid at position A14, A15,B1 (for insulin based B chains), B2 (for IGF-1 based B chains), B10,B22, B28 or B29 (according to the amino acid numbering of the A and Bchain of native insulin).

In some embodiments of the invention, the FGF21 based conjugatecomprises a spacer between the peptide and the alkyl group. In someembodiments, the FGF21 based conjugate is covalently bound to thespacer, which is covalently bound to the alkyl group. In some exemplaryembodiments, the FGF21 based conjugate is modified to comprise an alkylgroup by alkylation of an amine, hydroxyl, or thiol of a spacer, whereinthe spacer is attached to a side chain of an amino acid of theconjugate. The amino acid of the FGF21 based conjugate to which thespacer is attached can be any amino acid (e.g., a singly α-substitutedamino acid or an α,α-disubstituted amino acid) comprising a moiety whichpermits linkage to the spacer. An amino acid of the FGF21 basedconjugate comprising a side chain —NH₂, —OH, or —COOH (e.g., Lys, Orn,Ser, Asp, or Glu) is suitable. In some embodiments, the spacer betweenthe peptide the FGF21 based conjugate and the alkyl group is an aminoacid comprising a side chain amine, hydroxyl, or thiol or a dipeptide ortripeptide comprising an amino acid comprising a side chain amine,hydroxyl, or thiol.

In the instance in which the alpha amine is alkylated, the spacer aminoacid can be any amino acid. For example, the spacer amino acid can be ahydrophobic amino acid, e.g., Gly, Ala, Val, Leu, Ile, Trp, Met, Phe,Tyr. Alternatively, the spacer amino acid can be an acidic residue,e.g., Asp and Glu. In exemplary embodiments, the spacer amino acid canbe a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, Be, Trp, Met,Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoicacid, 8-aminooctanoic acid. Alternatively, the spacer amino acid can bean acidic residue, e.g., Asp and Glu, provided that the alkylationoccurs on the alpha amine of the acidic residue. In the instance inwhich the side chain amine of the spacer amino acid is alkylated, thespacer amino acid is an amino acid comprising a side chain amine, e.g.,an amino acid of Formula I (e.g., Lys or Orn). In this instance, it ispossible for both the alpha amine and the side chain amine of the spaceramino acid to be alkylated, such that the peptide is dialkylated.Embodiments of the invention include such dialkylated molecules.

In some embodiments, the spacer comprises a hydrophilic bifunctionalspacer. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.). In some embodiments, thespacer between peptide the FGF21 based conjugate and the alkyl group isa hydrophilic bifunctional spacer. In certain embodiments, thehydrophilic bifunctional spacer comprises two or more reactive groups,e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or anycombinations thereof. In certain embodiments, the hydrophilicbifunctional spacer comprises a hydroxyl group and a carboxylate. Inother embodiments, the hydrophilic bifunctional spacer comprises anamine group and a carboxylate. In other embodiments, the hydrophilicbifunctional spacer comprises a thiol group and a carboxylate.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional spacer, or hydrophobic bifunctional spacer) is 3 to 10atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or 10 atoms)) in length.In more specific embodiments, the spacer is about 3 to 10 atoms (e.g., 6to 10 atoms) in length and the alkyl is a C₁₂ to C₁₈ alkyl group, e.g.,C₁₄ alkyl group, C₁₆ alkyl group, such that the total length of thespacer and alkyl group is 14 to 28 atoms, e.g., about 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 atoms. In some embodimentsthe length of the spacer and alkyl is 17 to 28 (e.g., 19 to 26, 19 to21) atoms.

In accordance with one embodiment the bifunctional spacer is a syntheticor non-naturally occurring amino acid comprising an amino acid backbonethat is 3 to 10 atoms in length (e.g., 6-amino hexanoic acid,5-aminovaleric acid, 7-aminoheptanoic acid, and 8-aminooctanoic acid).Alternatively, the spacer can be a dipeptide or tripeptide spacer havinga peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) inlength. The dipeptide or tripeptide spacer attached to the FGF21 basedconjugate can be composed of naturally-occurring and/or non-naturallyoccurring amino acids, including, for example, any of the amino acidstaught herein. In some embodiments the spacer comprises an overallnegative charge, e.g., comprises one or two negatively charged aminoacids. In some embodiments the dipeptide spacer is selected from thegroup consisting of: Ala-Ala, β-Ala-β-Ala, Leu-Leu, Pro-Pro,γ-aminobutyric acid-γ-aminobutyric acid, and γ-Glu-γ-Glu. In oneembodiment the dipeptide spacer is γ-Glu-γ-Glu.

Suitable methods of peptide alkylation via amines, hydroxyls, and thiolsare known in the art. For example, a Williamson ether synthesis can beused to form an ether linkage between the insulin peptide and the alkylgroup. Also, a nucleophilic substitution reaction of the peptide with analkyl halide can result in any of an ether, thioether, or amino linkage.The alkyl group of the alkylated peptide the FGF21 based conjugate canbe of any size, e.g., any length carbon chain, and can be linear orbranched. In some embodiments of the invention, the alkyl group is a C₄to C₃₀ alkyl. For example, the alkyl group can be any of a C₄ alkyl, C₆alkyl, C₈ alkyl, C₁₀ alkyl, C₁₂ alkyl, C₁₄ alkyl, C₁₆ alkyl, C₁₈ alkyl,C₂₀ alkyl, C₂₂ alkyl, C₂₄ alkyl, C₂₆ alkyl, C₂₈ alkyl, or a C₃₀ alkyl.In some embodiments, the alkyl group is a C₈ to C₂₀ alkyl, e.g., a C₁₄alkyl or a C₁₆ alkyl.

In some specific embodiments, the alkyl group comprises a steroid moietyof a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholicacid, lithocholic acid, taurocholic acid, glycocholic acid, andcholesterol acid.

When a long chain alkane is alkylated by the FGF21 based conjugate orthe spacer, the long chain alkane may be of any size and can compriseany length of carbon chain. The long chain alkane can be linear orbranched. In certain aspects, the long chain alkane is a C₄ to C₃₀alkane. For example, the long chain alkane can be any of a C₄ alkane, C₆alkane, C₈ alkane, C₁₀ alkane, C₁₂ alkane, C₁₄ alkane, C₁₆ alkane, C₁₈alkane, C₂₀ alkane, C₂₂ alkane, C₂₄ alkane, C₂₆ alkane, C₂₈ alkane, or aC₃₀ alkane. In some embodiments the long chain alkane comprises a C₈ toC₂₀ alkane, e.g., a C₁₄ alkane, C₁₆ alkane, or a C₁₈ alkane.

Also, in some embodiments alkylation can occur between the insulinanalog and a cholesterol moiety. For example, the hydroxyl group ofcholesterol can displace a leaving group on the long chain alkane toform a cholesterol-insulin peptide product.

Self Cleaving Dipeptide Element

In accordance with one embodiment the insulin peptide of the conjugatesdisclosed herein are further modified to comprise a self cleavingdipeptide element. In one embodiment the dipeptide element comprises thestructure U-J, wherein U is an amino acid or a hydroxyl acid and J is anN-alkylated amino acid. In one embodiment one or more dipeptide elementsare linked to the FGF21 based insulin conjugate through an amide bondformed through one or more amino groups selected from the N-terminalamino group of the A or B chain of the insulin component, or the sidechain amino group of an amino acid present in the conjugate. Inaccordance with one embodiment one or more dipeptide elements are linkedto the FGF21 based insulin conjugate at an amino group selected from theN-terminal amino group of the conjugate, or the side chain amino groupof an aromatic amine of a 4-amino-phenylalanine residue present at aposition corresponding to position A19, B16 or B25 of native insulin, ora side chain of an amino acid of the linking moiety of a single chaininsulin analog.

In one embodiment the dipeptide prodrug element comprises the generalstructure of Formula X:

-   -   wherein    -   R₁, R₂, R₄ and R₈ are independently selected from the group        consisting of H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH,        (C₁-C₁₈ alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄        alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₂-C₅ heterocyclic),        (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄ alkyl)(C₃-C₉ heteroaryl),        and C₁-C₁₂ alkyl(W)C₁-C₁₂ alkyl, wherein W is a heteroatom        selected from the group consisting of N, S and O, or R₁ and R₂        together with the atoms to which they are attached form a C₃-C₁₂        cycloalkyl or aryl; or R₄ and R₈ together with the atoms to        which they are attached form a C₃-C₆ cycloalkyl;    -   R₃ is selected from the group consisting of C₁-C₁₈ alkyl,        (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄        alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic),        (C₀-C₄ alkyl)(C₆—C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉        heteroaryl) or R₄ and R₃ together with the atoms to which they        are attached form a 4, 5 or 6 member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, C₁-C₈ alkyl or R₆ and R₂ together with the atoms to        which they are attached form a 4, 5 or 6 member heterocyclic        ring; and    -   R₇ is selected from the group consisting of H and OH. In one        embodiment when the prodrug element is linked to the N-terminal        amine of the FGF21 based insulin conjugate and R₄ and R₃        together with the atoms to which they are attached form a 4, 5        or 6 member heterocyclic ring, then at least one of R₁ and R₂        are other than H.

In one embodiment a complex is provided comprising the general structureA-B-Y A-B-(Q-L-Y), wherein Y represents any of the FGF21 analogs asdescribed elsewhere in this disclosure, Q-L-Y comprises any of theconjugates as described elsewhere in this disclosure and A-B is adipeptide that is linked via an amide bond to an amine of Y or the Q-L-Yconjugate. In one embodiment A-B is linked to amine present on theinsulin peptide of an FGF21 analog insulin conjugate. In one embodimentA-B is linked to the N-terminal alpha amine of the A or B chain of theinsulin peptide of the FGF21 analog conjugate.

In one embodiment the dipeptide A-B (having the structure of Formula IV)is covalently linked to the alpha amine of an FGF21 analog comprisingthe sequence of SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 204, SEQ IDNO: 205, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250,SEQ ID NO: 251 and SEQ ID NO: 252.

In one embodiment, a complex of the structure A-B-(Q-L-Y) is provided,wherein Q-L-Y comprises any of the structures as described elsewhere inthis disclosure and wherein

A is an amino acid or a hydroxy acid;

B is an N-alkylated amino acid linked to Q through an amide bond betweena carboxyl moiety of B and an amine of Q; and

A-B comprises the structure:

wherein

-   -   (a) R¹, R², R⁴ and R⁸ are independently selected from the group        consisting of H, C1-C18 alkyl, C2-C18 alkenyl, (C1-C18 alkyl)OH,        (C1-C18 alkyl)SH, (C2-C3 alkyl)SCH₃, (C1-C4 alkyl)CONH₂, (C1-C4        alkyl)COOH, (C1-C4 alkyl)NH₂, (C1-C4 alkyl)NHC(NH₂ ⁺)NH₂, (C0-C4        alkyl)(C3-C6 cycloalkyl), (C0-C4 alkyl)(C2-C5 heterocyclic),        (C0-C4 alkyl)(C6-C10 aryl)R⁷, (C1-C4 alkyl)(C3-C9 heteroaryl),        and C1-C12 alkyl(W1)C1-C12 alkyl, wherein W1 is a heteroatom        selected from the group consisting of N, S and O, or        -   (ii) R¹ and R² together with the atoms to which they are            attached form a C3-C12 cycloalkyl or aryl; or        -   (iii) R⁴ and R⁸ together with the atoms to which they are            attached form a C3-C6 cycloalkyl;    -   (b) R³ is selected from the group consisting of C1-C18 alkyl,        (C1-C18 alkyl)OH, (C1-C18 alkyl)NH₂, (C1-C18 alkyl)SH, (C0-C4        alkyl)(C3-C6)cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocyclic),        (C0-C4 alkyl)(C6-C10 aryl)R⁷, and (C1-C4 alkyl)(C3-C9        heteroaryl) or R⁴ and R³ together with the atoms to which they        are attached form a 4, 5 or 6 member heterocyclic ring;    -   (c) R⁵ is NHR⁶ or OH;    -   (d) R⁶ is H, C₁-C₈ alkyl; and    -   (e) R⁷ is selected from the group consisting of H and OH

wherein the chemical cleavage half-life (t_(1/2)) of A-B from Q or Y isat least about 1 hour to about 1 week in PBS under physiologicalconditions.

In a further embodiment, A-B comprises the structure:

wherein

-   -   R₁ and R₈ are independently H or C₁-C₈ alkyl;    -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃        alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄        alkyl)NH₂, and (C₁-C₄ alkyl)(C₆ aryl)R₇;    -   R₃ is C₁-C₆ alkyl;    -   R₅ is NH₂; and    -   R₇ is selected from the group consisting of hydrogen, and OH.

In a further embodiment, A-B comprises the structure:

wherein

-   -   R₁ is H;    -   R₂ is H, C₁-C₄ alkyl, (CH₂ alkyl)OH, (C₁-C₄ alkyl)NH₂, or        (CH₂)(C₆ aryl)R₇;    -   R₃ is C₁-C₆ alkyl;    -   R₄ is H, C₁-C₄ alkyl, or (CH₂)(C₆ aryl)R₇;    -   R₅ is NH₂;    -   R₈ is hydrogen; and    -   R₇ is H or OH.

In a further embodiment, A-B comprises the structure:

wherein

-   -   R₁ is H or C₁-C₄ alkyl;    -   R₂ is H, C₁-C₄ alkyl, or (C₁-C₄ alkyl)NH₂;    -   R₃ is C₁-C₆ alkyl;    -   R₄ is H, or C₁-C₄ alkyl;    -   R₅ is NH₂; and    -   R₈ is hydrogen.

Pharmaceutical compositions comprising the FGF21 based conjugatesdisclosed herein can be formulated and administered to patients usingstandard pharmaceutically acceptable carriers and routes ofadministration known to those skilled in the art. Accordingly, thepresent disclosure also encompasses pharmaceutical compositionscomprising one or more of the FGF21 based conjugates disclosed herein ora pharmaceutically acceptable salt thereof, in combination with apharmaceutically acceptable carrier. In one embodiment thepharmaceutical composition comprises a 1 mg/ml concentration of theFGF21 based conjugate at a pH of about 4.0 to about 7.0 in a phosphatebuffer system. The pharmaceutical compositions may comprise the FGF21based conjugate as the sole pharmaceutically active component, or theFGF21 based conjugate peptide can be combined with one or moreadditional active agents.

All therapeutic methods, pharmaceutical compositions, kits and othersimilar embodiments described herein contemplate that FGF21 basedconjugate peptides include all pharmaceutically acceptable saltsthereof.

In one embodiment the kit is provided with a device for administeringthe FGF21 based conjugate to a patient. The kit may further include avariety of containers, e.g., vials, tubes, bottles, and the like.Preferably, the kits will also include instructions for use. Inaccordance with one embodiment the device of the kit is an aerosoldispensing device, wherein the composition is prepackaged within theaerosol device. In another embodiment the kit comprises a syringe and aneedle, and in one embodiment the FGF21 based conjugate composition isprepackaged within the syringe.

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins. Although certain non-natural amino acidscannot be expressed by standard recombinant DNA techniques, techniquesfor their preparation are known in the art. Compounds of this inventionthat encompass non-peptide portions may be synthesized by standardorganic chemistry reactions, in addition to standard peptide chemistryreactions when applicable.

Exemplary Embodiments Embodiment 1

A peptide exhibiting antagonist activity against Klotho (3, said peptidecomprising an amino acid sequence ofX₁X₂X₃X₄X₅SX₇DPX₁₀X₁₁X₁₂VX₁₄GX₁₆X₁₇X₁₈X₁₉RSPSX₂₄X₂₅X₂₆ (SEQ ID NO: 235),

wherein

X₁ is Pro or absent;

X₂ is Pro or Leu;

X₃ is Asp or Glu;

X₄ is Val or Thr;

X₅ is Gly, Asp, Phe, Leu or Ser;

X₇ is Ser or Met;

X₁₀ is Leu or Phe;

X₁₁ is Ser or Gly;

X₁₂ is Met or Leu;

X₁₄ is absent or Thr;

X₁₆ is Pro, Leu, Arg, Glu, or Gly;

X₁₇ is Ser or Glu;

X₁₈ is Gln or Ala;

X₁₉ is Gly or Val;

X₂₄ is Tyr or Phe;

X₂₅ is Ala or Glu; and

X₂₆ is an aliphatic amino acid selected from Gly, Ala, Val, Leu, Ser, orIle, optionally comprising up to 5 further amino acid substitutions.

Embodiment 2

The peptide according to embodiment 1, wherein the peptide of SEQ ID NO:235 comprises up to 4 further amino acid substitutions.

Embodiment 3

The peptide according to any one of the preceding embodiments whereinthe peptide of SEQ ID NO: 235 comprises up to 3 further amino acidsubstitutions.

Embodiment 4

The peptide according to any one of the preceding embodiments whereinthe peptide of SEQ ID NO: 235 comprises up to 2 further amino acidsubstitutions.

Embodiment 5

The peptide according to any one of the preceding embodiments whereinthe peptide of SEQ ID NO: 235 comprises up to 1 further amino acidsubstitutions.

Embodiment 6

The peptide according to any one of the preceding embodiments comprisingthe sequence wherein

X₁X₂X₃X₄X₅SX₇DPX₁₀X₁₁X₁₂VX₁₄GX₁₆X₁₇X₁₈X₁₉RSPSX₂₄X₂₅X₂₆ (SEQ ID NO: 235),

wherein

X₁ is Pro or absent;

X₂ is Pro or Leu;

X₃ is Asp or Glu;

X₄ is Val or Thr;

X₅ is Gly, Asp, Phe, Leu or Ser;

X₇ is Ser or Met;

X₁₀ is Leu or Phe;

X₁₁ is Ser or Gly;

X₁₂ is Met or Leu;

X₁₄ is absent or Thr;

X₁₆ is Pro, Leu, or Arg;

X₁₇ is Ser or Glu;

X₁₈ is Gln or Ala;

X₁₉ is Gly or Val;

X₂₄ is Tyr or Phe;

X₂₅ is Ala or Glu; and

X₂₆ is an aliphatic amino acid selected from Gly, Ala, Val, Leu, Ser, orIle.

Embodiment 7

The peptide according to any one of the preceding embodiments comprisingthe sequence X₁X₂X₃X₄X₅SX₇DPX₁₀X₁₁X₁₂VX₁₄GX₁₆X₁₇X₁₈X₁₉RSPSX₂₄X₂₅A (SEQID NO: 236),

wherein

X₁ is Pro or absent;

X₂ is Pro or Leu;

X₃ is Asp or Glu;

X₄ is Val or Thr;

X₅ is Gly, Asp, Phe, Leu or Ser;

X₇ is Ser or Met;

X₁₀ is Leu or Phe;

X₁₁ is Ser or Gly;

X₁₂ is Met or Leu;

X₁₄ is absent or Thr;

X₁₆ is Pro, Leu, or Arg;

X₁₇ is Ser or Glu;

X₁₈ is Gln or Ala;

X₁₉ is Gly or Val;

X₂₄ is Tyr or Phe; and

X₂₅ is Ala or Glu.

Embodiment 8

The peptide according to any one of the preceding embodiments wherein X₁is Pro.

Embodiment 9

The peptide according to any one of the preceding embodiments wherein X₁is absent.

Embodiment 10

The peptide according to any one of the preceding embodiments wherein X₂is Pro.

Embodiment 11

The peptide according to any one of the preceding embodiments wherein X₂is Leu.

Embodiment 12

The peptide according to any one of the preceding embodiments wherein X₃is Asp.

Embodiment 13

The peptide according to any one of the preceding embodiments wherein X₃is Glu.

Embodiment 14

The peptide according to any one of the preceding embodiments wherein X₄is Val.

Embodiment 15

The peptide according to any one of the preceding embodiments wherein X₄is Thr.

Embodiment 16

The peptide according to any one of the preceding embodiments wherein X₅is Gly.

Embodiment 17

The peptide according to any one of the preceding embodiments wherein X₅is Asp.

Embodiment 18

The peptide according to any one of the preceding embodiments wherein X₅is Phe.

Embodiment 19

The peptide according to any one of the preceding embodiments wherein X₅is Leu.

Embodiment 20

The peptide according to any one of the preceding embodiments wherein X₅is Ser.

Embodiment 21

The peptide according to any one of the preceding embodiments wherein X₇is Ser.

Embodiment 22

The peptide according to any one of the preceding embodiments wherein X₇is Met.

Embodiment 23

The peptide according to any one of the preceding embodiments whereinX₁₀ is Leu.

Embodiment 24

The peptide according to any one of the preceding embodiments whereinX₁₀ is Phe.

Embodiment 25

The peptide according to any one of the preceding embodiments whereinX₁₁ is Ser.

Embodiment 26

The peptide according to any one of the preceding embodiments whereinX₁₁ is Gly.

Embodiment 27

The peptide according to any one of the preceding embodiments whereinX₁₂ is Met.

Embodiment 28

The peptide according to any one of the preceding embodiments whereinX₁₂ is Leu.

Embodiment 29

The peptide according to any one of the preceding embodiments whereinX₁₄ is absent.

Embodiment 30

The peptide according to any one of the preceding embodiments whereinX₁₄ is Thr.

Embodiment 31

The peptide according to any one of the preceding embodiments whereinX₁₆ is Pro.

Embodiment 32

The peptide according to any one of the preceding embodiments whereinX₁₆ is Leu.

Embodiment 33

The peptide according to any one of the preceding embodiments whereinX₁₆ is Arg.

Embodiment 34

The peptide according to any one of the preceding embodiments whereinX₁₆ is Glu.

Embodiment 35

The peptide according to any one of the preceding embodiments whereinX₁₆ is Gly.

Embodiment 36

The peptide according to any one of the preceding embodiments whereinX₁₇ is Ser.

Embodiment 37

The peptide according to any one of the preceding embodiments whereinX₁₇ is Glu.

Embodiment 38

The peptide according to any one of the preceding embodiments whereinX₁₈ is Gln.

Embodiment 39

The peptide according to any one of the preceding embodiments whereinX₁₈ is Ala.

Embodiment 40

The peptide according to any one of the preceding embodiments whereinX₁₉ is Gly.

Embodiment 41

The peptide according to any one of the preceding embodiments whereinX₁₉ is Val.

Embodiment 42

The peptide according to any one of the preceding embodiments whereinX₂₄ is Tyr.

Embodiment 43

The peptide according to any one of the preceding embodiments whereinX₂₄ is Phe.

Embodiment 44

The peptide according to any one of the preceding embodiments whereinX₂₅ is Ala.

Embodiment 45

The peptide according to any one of the preceding embodiments whereinX₂₅ is Glu.

Embodiment 46

The peptide according to any one of the preceding embodiments whereinX₂₆ is Gly.

Embodiment 47

The peptide according to any one of the preceding embodiments whereinX₂₆ is Ala.

Embodiment 48

The peptide according to any one of the preceding embodiments whereinX₂₆ is Val.

Embodiment 49

The peptide according to any one of the preceding embodiments whereinX₂₆ is Leu.

Embodiment 50

The peptide according to any one of the preceding embodiments whereinX₂₆ is Ser.

Embodiment 51

The peptide according to any one of the preceding embodiments whereinX₂₆ is Be.

Embodiment 52

The peptide according to any one of the preceding embodiments wherein

X₁ is Pro or absent;

X₂ is Pro or Leu; X₃ is Asp or Glu; X₄ is Val or Thr; X₅ is Gly or Asp;X₇ is Ser or Met; X₁₀ is Leu or Phe; X₁₁ is Ser or Gly; X₁₂ is Met orLeu;

X₁₄ is absent or Thr;

X₁₆ is Pro or Leu; X₁₇ is Ser or Glu; X₁₈ is Gln or Ala; X₁₉ is Gly orVal; X₂₄ is Tyr or Phe; and X₂₅ is Ala or Glu. Embodiment 53

The peptide according to any one of the preceding embodiments wherein

X₁ is Pro; X₂ is Pro; X₃ is Asp; X₄ is Val; X₁₀ is Phe; X₁₁ is Gly; X₁₂is Leu;

X₁₄ is absent;

X₁₆ is Pro; X₁₇ is Ser; X₁₈ is Gln; X₁₉ is Gly; X₂₄ is Phe and X₂₅ isGlu. Embodiment 54

The peptide according to any one of the preceding embodiments comprisinga sequence selected from the group consisting of

PPDVG SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 180), PPDVG SMDPF GLVGR SQGRSPSFEA (0470; SEQ ID NO: 237), PPDVF SMDPF GLVGP SQGRS PSFEA (0480; SEQID NO: 238), PPDVL SMDPF GLVGP SQGRS PSFEA (0481; SEQ ID NO: 239), PPDVSSMDPF GLVGP SQGRS PSFEA (0482; SEQ ID NO: 240), PPDVG SSDPF GLVGP SQGRSPSFEA (0486; SEQ ID NO: 241), PPDVG SSDPL SMVGP SQGRS PSYAA (FGF21 C25;SEQ ID NO: 191), PLETD SMDPF GLVTG LEAVR SPSFE A (FGF19 A25; SEQ ID NO:178), PLETD SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 179), PPDVG SMDPF GLVTGLEAVR SPSYA A (SEQ ID NO: 182), PPDVG SSDPL SMVTG LEAVR SPSFE A (0435;SEQ ID NO: 184), and LETDS MDPFG LVTGL EAVRS PSFEA (SEQ ID NO: 188).Embodiment 55

The peptide according to any one of the preceding embodiments comprisinga sequence selected from the group consisting of

PPDVG SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 180), PPDVG SMDPF GLVGR SQGRSPSFEA (0470; SEQ ID NO: 237), PPDVF SMDPF GLVGP SQGRS PSFEA (0480; SEQID NO: 238), PPDVL SMDPF GLVGP SQGRS PSFEA (0481; SEQ ID NO: 239), PPDVSSMDPF GLVGP SQGRS PSFEA (0482; SEQ ID NO: 240), and PPDVG SSDPF GLVGPSQGRS PSFEA (0486; SEQ ID NO: 241). Embodiment 56

The peptide according to any one of the preceding embodiments comprisinga sequence selected from the group consisting of

PLETD SMDPF GLVTG LEAVR SPSFE A (FGF19 A25; SEQ ID NO: 178), PLETD SMDPFGLVGP SQGRS PSFEA (SEQ ID NO: 179), and LETDS MDPFG LVTGL EAVRS PSFEA(SEQ ID NO: 188). Embodiment 57

The peptide according to any one of the preceding embodiments comprisingthe sequence

PPDVG SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 180). Embodiment 58

The peptide according to any one of the preceding embodiments comprisingthe sequence

PPDVG SMDPF GLVGR SQGRS PSFEA (0470; SEQ ID NO: 237). Embodiment 59

The peptide according to any one of the preceding embodiments comprisingthe sequence

PPDVF SMDPF GLVGP SQGRS PSFEA (0480; SEQ ID NO: 238). Embodiment 60

The peptide according to any one of the preceding embodiments comprisingthe sequence

PPDVL SMDPF GLVGP SQGRS PSFEA (0481; SEQ ID NO: 239). Embodiment 61

The peptide according to any one of the preceding embodiments comprisingthe sequence

PPDVS SMDPF GLVGP SQGRS PSFEA (0482; SEQ ID NO: 240). Embodiment 62

The peptide according to any one of the preceding embodiments comprisingthe sequence

PPDVG SSDPF GLVGP SQGRS PSFEA (0486; SEQ ID NO: 241). Embodiment 63

The peptide according to any one of the preceding embodiments comprisingthe sequence

PPDVG SSDPL SMVGP SQGRS PSYAA (FGF21 C25; SEQ ID NO: 191). Embodiment 64

The peptide according to any one of the preceding embodiments comprisingthe sequence

PLETD SMDPF GLVTG LEAVR SPSFE A (FGF19 A25; SEQ ID NO: 178). Embodiment65

The peptide according to any one of the preceding embodiments comprisingthe sequence

PLETD SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 179). Embodiment 66

The peptide according to any one of the preceding embodiments comprisingthe sequence

PPDVG SMDPF GLVTG LEAVR SPSYA A (SEQ ID NO: 182). Embodiment 67

The peptide according to any one of the preceding embodiments comprisingthe sequence

PPDVGSSDPLSMVTGLEAVRSPSFE A (0435; SEQ ID NO: 184). Embodiment 68

The peptide according to any one of the preceding embodiments comprisingthe sequence

LETDS MDPFG LVTGL EAVRS PSFEA (SEQ ID NO: 188). Embodiment 69

An FGF21 peptide comprising the structure of A-B wherein A is a peptideaccording to SEQ ID NO: 195, optionally comprising up to 10 furtheramino acid modifications, and B is a peptide of any one of embodiments 1to 68.

Embodiment 70

The FGF21 peptide according to embodiment 69 wherein A optionallycomprises up to 9 further amino acid modifications.

Embodiment 71

The FGF21 peptide according to embodiment 69 wherein A optionallycomprises up to 8 further amino acid modifications.

Embodiment 72

The FGF21 peptide according to embodiment 69 wherein A optionallycomprises up to 7 further amino acid modifications.

Embodiment 73

The FGF21 peptide according to embodiment 69 wherein A optionallycomprises up to 6 further amino acid modifications.

Embodiment 74

The FGF21 peptide according to embodiment 69 wherein A optionallycomprises up to 5 further amino acid modifications.

Embodiment 75

The FGF21 peptide according to embodiment 69 wherein A optionallycomprises up to 4 further amino acid modifications.

Embodiment 76

The FGF21 peptide according to embodiment 69 wherein A optionallycomprises up to 3 further amino acid modifications.

Embodiment 77

The FGF21 peptide according to embodiment 69 wherein A optionallycomprises up to 2 further amino acid modifications.

Embodiment 78

The FGF21 peptide according to embodiment 69 wherein A optionallycomprises 1 further amino acid modification.

Embodiment 79

The FGF21 peptide according to any one of embodiments 69 to 78 wherein Ais a peptide according to SEQ ID NO: 194.

Embodiment 80

The FGF21 peptide according to any one of embodiments 69 to 78 wherein Acomprises one or more of amino acid modifications A31C, G43C, L98D,L100K, N121D, and/or D127K.

Embodiment 81

The FGF21 peptide according to any one of embodiments 69 to 78 wherein Acomprises the amino acid modifications A31C, G43C, L98D, L100K, N121D,and D127K (SEQ ID NO: 196).

Embodiment 82

The FGF21 peptide according to any one of embodiments 69 to 78 wherein Ais a peptide according to SEQ ID NO: 195.

Embodiment 83

The FGF21 peptide according to any one of embodiments 69 to 78 wherein Ais a peptide according to SEQ ID NO: 195 and B is selected from the listconsisting of

PPDVG SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 180) PPDVG SMDPF GLVGR SQGRSPSFEA (0470; SEQ ID NO: 237), PPDVF SMDPF GLVGP SQGRS PSFEA (0480; SEQID NO: 238), PPDVL SMDPF GLVGP SQGRS PSFEA (0481; SEQ ID NO: 239), PPDVSSMDPF GLVGP SQGRS PSFEA (0482; SEQ ID NO: 240), PPDVG SSDPF GLVGP SQGRSPSFEA (0486; SEQ ID NO: 241), PPDVG SSDPL SMVGP SQGRS PSYAA (FGF21 C25;SEQ ID NO: 191), PLETD SMDPF GLVTG LEAVR SPSFE A (FGF19 A25; SEQ ID NO:178), PLETD SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 179), PPDVG SMDPF GLVTGLEAVR SPSYA A (SEQ ID NO: 182), PPDVG SSDPL SMVTG LEAVR SPSFE A (0435;SEQ ID NO: 184), and LETDS MDPFG LVTGL EAVRS PSFEA (SEQ ID NO: 188).Embodiment 84

The FGF21 peptide according to any one of embodiments 69 to 78 wherein Ais a peptide according to SEQ ID NO: 195 and B is selected from thegroup consisting of

PPDVG SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 180), PPDVG SMDPF GLVGR SQGRSPSFEA (0470; SEQ ID NO: 237), PPDVF SMDPF GLVGP SQGRS PSFEA (0480; SEQID NO: 238), PPDVL SMDPF GLVGP SQGRS PSFEA (0481; SEQ ID NO: 239), PPDVSSMDPF GLVGP SQGRS PSFEA (0482; SEQ ID NO: 240), and PPDVG SSDPF GLVGPSQGRS PSFEA (0486; SEQ ID NO: 241). Embodiment 85

The FGF21 peptide according to any one of embodiments 69 to 78 wherein

A is a peptide according to SEQ ID NO: 195 andB is selected from the group consisting of

PLETD SMDPF GLVTG LEAVR SPSFE A (FGF19 A25; SEQ ID NO: 178)PLETDSMDPFGLVGPSQGRSPSFEA (0430; SEQ ID NO: 179) LETDS MDPFG LVTGL EAVRSPSFEA (SEQ ID NO: 188) Embodiment 86

The FGF21 peptide according to any one of embodiments 69 to 78 wherein

A is a peptide according to SEQ ID NO: 195 and

B is LETDS MDPFG LVTGL EAVRS PSFEA (SEQ ID NO: 188). Embodiment 87

The FGF21 peptide according to any one of embodiments 69 to 78 wherein

A is a peptide according to SEQ ID NO: 196 andB is selected from the list consisting of

PPDVG SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 180) PPDVG SMDPF GLVGR SQGRSPSFEA (0470; SEQ ID NO: 237), PPDVF SMDPF GLVGP SQGRS PSFEA (0480; SEQID NO: 238), PPDVL SMDPF GLVGP SQGRS PSFEA (0481; SEQ ID NO: 239), PPDVSSMDPF GLVGP SQGRS PSFEA (0482; SEQ ID NO: 240), PPDVG SSDPF GLVGP SQGRSPSFEA (0486; SEQ ID NO: 241), PPDVG SSDPL SMVGP SQGRS PSYAA (FGF21 C25;SEQ ID NO: 191), PLETD SMDPF GLVTG LEAVR SPSFE A (FGF19 A25; SEQ ID NO:178), PLETD SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 179), PPDVG SMDPF GLVTGLEAVR SPSYA A (SEQ ID NO: 182), PPDVGSSDPLSMVTGLEAVRSPSFE A (0435; SEQID NO: 184), and LETDS MDPFG LVTGL EAVRS PSFEA (SEQ ID NO: 188)Embodiment 88

The FGF21 peptide according to any one of embodiments 69 to 78 wherein

A is a peptide according to SEQ ID NO: 196 andB is selected from the group consisting of

PPDVG SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 180) PPDVG SMDPF GLVGR SQGRSPSFEA (0470; SEQ ID NO: 237), PPDVF SMDPF GLVGP SQGRS PSFEA (0480; SEQID NO: 238), PPDVL SMDPF GLVGP SQGRS PSFEA (0481; SEQ ID NO: 239), PPDVSSMDPF GLVGP SQGRS PSFEA (0482; SEQ ID NO: 240), and PPDVG SSDPF GLVGPSQGRS PSFEA (0486; SEQ ID NO: 241). Embodiment 89

The FGF21 peptide according to any one of embodiments 69 to 78 wherein

A is a peptide according to SEQ ID NO: 196 andB is selected from the group consisting of

PLETD SMDPF GLVTG LEAVR SPSFE A (FGF19 A25; SEQ ID NO: 178), PLETD SMDPFGLVGP SQGRS PSFEA (SEQ ID NO: 179), and LETDS MDPFG LVTGL EAVRS PSFEA(SEQ ID NO: 188). Embodiment 90

The FGF21 peptide according to any one of embodiments 69 to 78 wherein

A is a peptide according to SEQ ID NO: 196 and

B is LETDS MDPFG LVTGL EAVRS PSFEA (SEQ ID NO: 188). Embodiment 91

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of SEQ ID NO: 192.

Embodiment 92

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of SEQ ID NO: 193.

Embodiment 93

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of SEQ ID NO: 206.

Embodiment 94

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of SEQ ID NO: 207.

Embodiment 95

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of SEQ ID NO: 208.

Embodiment 96

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of SEQ ID NO: 209.

Embodiment 97

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of the sequence

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSMDPFGLVGRS QGRSPSFEA(0470; SEQ ID NO: 242). Embodiment 98

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of the sequence

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVFSMDPFGLVGPSQ GRSPSFEA(0480; SEQ ID NO: 243). Embodiment 99

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of the sequence

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVLSMDPFGLVGPS Q GRSPSFEA(0481; SEQ ID NO: 244). Embodiment 100

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of the sequence

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVSSMDPFGLVGPSQ GRSPSFEA(0482; SEQ ID NO: 245). Embodiment 101

The FGF21 peptide according to any one of embodiments 69 to 90 whereinthe peptide consists of the sequence

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPFGLVGPSQ GRSPSFEA(0486; SEQ ID NO: 246). Embodiment 102

A pharmaceutical composition comprising the FGF21 peptide according toany one of embodiments 69 to 101 and pharmaceutically acceptableexcipient.

Embodiment 103

The FGF21 peptide according to any one of embodiments 69 to 101 for useas a medicament.

Embodiment 104

The FGF21 peptide according to any one of embodiments 69 to 101 for usein the treatment of diabetes.

Embodiment 105

The FGF21 peptide according to any one of embodiments 69 to 101 for usein the treatment of obesity.

Embodiment 106

The FGF21 peptide according to any one of embodiments 69 to 101 for usein reducing weight gain and inducing weight loss.

Example 1

Fibroblast growth factor-21 (FGF21) has been intensively studied as ametabolic hormone with a particular interest in its therapeuticpotential. At the cellular level, FGF21 interacts with a complex offibroblast growth factor receptor (FGFR) and a tissue specificco-receptor Klotho β (KLB). The N- and C-termini of FGF21 are vital foreffective biochemical signaling. The deletion of the seventeenN-terminal residues of FGF21 (FGF21 18-181) inactivates the molecule togenerate a competitive antagonist of the native hormone. Here, we havedemonstrated that the C-terminal fragment of FGF21 as well as FGF19 arecapable of fully antagonizing the native FGF21 in vitro signaling.

Materials & Methods

Protein Synthesis:

The human FGF21 or FGF19 gene sequence was inserted in modifiedexpression pET21b vector containing yeast small ubiquitin-like modifier(SUMO) sequence after 6×His tag, using In-Fusion HD EcoDry Cloning Pluskit. For the generation of point mutant analogs, corresponding primerswere obtained from Integrated DNA technologies and mutagenesis wasperformed by standard PCR method. E. coli OrigamiB(DE3) cells weretransformed with modified pET vector containing the gene of interestfused to a His₆-sumo tag. FGF21 protein expression was induced overnightand the cells were harvested. The soluble whole cell lysate was appliedto a nickel affinity chromatography column for enrichment of the desiredprotein. Subsequently, the tag was cleaved and pure protein was obtainedby anion exchange chromatography.

Cell Culture:

Cells were cultured in 10% Fetal Bovine Serum containing DMEM Highglucose GlutaMAX at 37 r, 95% humidity, 5% CO₂. For generating the cellline with stable expression, human KLB gene was synthesized by Genscriptand subcloned into pcDNA3.1(+) with Zeocin resistance vector(Invitrogen) by NheI and NotI restriction enzyme sites. HEK 293T cellswere obtained from ATCC and were transiently transfected at 80%confluency using Lipofectamine 3000 (Invitrogen). Selection forKLB-expressing cells was initiated 48 hours post-transfection in thegrowth media containing 100 μg/ml of Zeocin (Gibco) and continued for 4weeks with fresh media added every third day. Human KLB expression inpooled cells was confirmed by Western blot and a functional FGF21 MAPKphosphorylation assay.

Erk1/2 Phosphorylation Assay:

293 HEK cells expressing hKLB were plated to 90% confluency in 96 wellplates coated with poly-D-Lysine. Cells were serum starved for threehours in 0.1% BSA containing media prior to stimulation with proteinand/or an antagonist peptide for ten minutes at 37° C. The cell lysatewas used for the detection of phospho-Erk1/2 levels by AlphaSureFire kitusing the prescribed protocol. The degree of biochemical activation wasrecorded and analyzed using Origin software by logistic curve fitting.Tests at each concentration were done in triplicates, and the standarddeviation is as shown in the graphs. The calculation of maximalactivities was done keeping the FGF21 157-181 or FGF19 169-194 peptideactivity as standard. The difference between the Erk1/2 phosphorylationsignal for the highest (10 μM) and the lowest (0 μM) tested dose fornative peptide was considered as 100% and accordingly all the othervalues were assigned.

Peptide Synthesis:

Synthesis was achieved using a Chemmatrix Rink amide resin using anautomated ABI433A or Symphony peptide synthesizer that employedFmoc/HOBT/DIC coupling protocols. The peptides were cleaved from thesolid-support using TFA/TIS/H₂O (95:2.5:2.5) for two hours. Followingether precipitation the peptide was solubilized in 20% CH₃CN andlyophilized. These peptides were purified by Waters Symmetry PreparativeC8 column with a linear gradient from aqueous CAN in 0.1% TFA. Thepurity within the set of site-specific alanine mutated peptides wasassessed by LCMS and concentrations were adjusted accordingly for the invitro assay.

Circular Dichroism:

The CD properties of FGF-proteins were recorded using a Jasco J-715instrument. The mean residue ellipticity was calculated and plotted as afunction of wavelength using Origin software.

Results

We observed that the C-terminal portion of FGF 19 or 21 can antagonizenative FGF21 signaling in similar fashion to the known, and much longerfragment 18-181. (see FIG. 1). The minimum effective length for FGF21antagonism was determined to be 25 amino acids, representing residues157-181 of the FGF21 protein (SEQ ID NO: 2), see the data presented inFIGS. 2 & 3A. The corresponding 26 residues of FGF19 (169-194) (FIG. 3B)were sufficient in replicating the effect that FGF21 18-181 elicits invitro. Both FGF21 and FGF19 C-terminal peptides were equally effectiveantagonists of FGF21 activity, suggesting that these peptides interactwith the receptor complex with same ability. Peptides shorter than 23amino acids in length were found to be non-functional. A representativeexample of non-functional smaller fragments is demonstrated by the FGF21162-181 peptide (FIG. 2). FGF21 159-181 peptide (23-amino acid)demonstrated weak antagonism and further extension by one residue toFGF21 160-181 peptide (24-amino acid) dramatically improved itsantagonistic potential, attaining the optimal performance at 25-aminoacid length. Peptides beyond the 25-mer were equally effectiveantagonists, not better, hence all further analyses were carried outwith the 25-mer peptide as the standard. It was confirmed that thephenomenon was KLB-receptor complex specific, since these peptides couldnot antagonize FGF1 activity in a similar setup for FGF23 inKL-expressing cells.

To gain more insight into the specific amino acid requirements for eachpeptide antagonist, we performed a complete alanine scan of the FGF21157-181 peptide and identified several sites with significantly alteredantagonistic activity (FIGS. 3A & 4). More particularly, Table 1provides a summary of the complete alanine scan of FGF21 157-181 andFGF19 169-194 peptide sequences. Each peptide was tested for its abilityto antagonize native FGF21 response by measuring the change inphosphorylation status of Erk1/2. The efficacy of each peptide isrepresented as % maximal activity, with native sequence attaining 100%response.

Table 2(A) lists a subset of peptides that achieve 95% or greatermaximal activity. Table 2(B) lists a subset of peptides that were notable to achieve a full response shown with their corresponding % maximalactivity. NC: Not calculated by the logistic curve fitting used. Thepeptides of Group A in Table 2(A) depicts the set of peptides thatshowed complete antagonism to the native FGF21 signaling and thusrepresent amino acids positions that were tolerant to the individualalanine substitutions, with some having marginally improved or worsepotencies with one exception; FGF19 169-194 K194A. Group B in Table 2(B)represents the peptides had a profound deleterious impact on theirantagonistic ability, such that they were unable to achieve fullantagonist response to native FGF21. The listed peptides of Table 2(B)were significantly weaker antagonists or inactive (maximal activity<95%and/or IC-50 values>1 μM).

TABLE 1 Alanine Scan of FGF21 25mer Modifi- IC-50 % max Peptide cation(nM) activity PPDVG SSDPL SMVGP SQGRS PSYAS (SEQ ID NO: 191) C25 168 ±83 100.0 APDVG SSDPL SMVGP SQGRS PSYAS (SEQ ID NO: 210) A1   33.0 100PADVG SSDPL SMVGP SQGRS PSYAS (SEQ ID NO: 211) A2  137.3  98.5PPAVG SSDPL SMVGP SQGRS PSYAS (SEQ ID NO: 212) A3 2283.2  87.8PPDAG SSDPL SMVGP SQGRS PSYAS (SEQ ID NO: 213) A4 5926.4  63.6PPDVA SSDPL SMVGP SQGRS PSYAS (SEQ ID NO: 214) A5   90.1 102.5PPDVG ASDPL SMVGP SQGRS PSYAS (SEQ ID NO: 215) A6 2647.4  89.2PPDVG SADPL SMVGP SQGRS PSYAS (SEQ ID NO: 216) A7  359.2  94.5PPDVG SSAPL SMVGP SQGRS PSYAS (SEQ ID NO: 217) A8PPDVG SSAPL SMVGP SQGRS PSYAS (SEQ ID NO: 218) A9PPDVG SSDPA SMVGP SQGRS PSYAS (SEQ ID NO: 219) A10 3315.9  76.0PPDVG SSDPL AMVGP SQGRS PSYAS (SEQ ID NO: 220) A11  101.6  98.0PPDVG SSDPL SAVGP SQGRS PSYAS (SEQ ID NO: 221) A12PPDVG SSDPL SMAGP SQGRS PSYAS (SEQ ID NO: 222) A13 2106.1  87.9PPDVG SSDPL SMVAP SQGRS PSYAS (SEQ ID NO: 223) A14  135.6  97.7PPDVG SSDPL SMVGA SQGRS PSYAS (SEQ ID NO: 224) A15  134.3  93.9PPDVG SSDPL SMVGP AQGRS PSYAS (SEQ ID NO: 225) A16  120.3 101.4PPDVG SSDPL SMVGP SAGRS PSYAS (SEQ ID NO: 226) A17  138.9  99.7PPDVG SSDPL SMVGP SQARS PSYAS (SEQ ID NO: 227) A18  139.0 102.9PPDVG SSDPL SMVGP SQGAS PSYAS (SEQ ID NO: 228) A19  102.1 100.1PPDVG SSDPL SMVGP SQGRA PSYAS (SEQ ID NO: 229) A20 3228.4  73.5PPDVG SSDPL SMVGP SQGRS ASYAS (SEQ ID NO: 230) A21 2242.5  70.5PPDVG SSDPL SMVGP SQGRS PAYAS (SEQ ID NO: 231) A22  752.1  78.5PPDVG SSDPL SMVGP SQGRS PSAAS (SEQ ID NO: 232) A23 3984.2  73.6PPDVG SSDPL SMVGP SQGRS PSYAA (SEQ ID NO: 233) A25   45.0  99.0

TABLE 2(A) ≥95% Maximal activity FGF21 FGF19 Position IC-50 (μM)Position IC-50 (μM) WT 0.16 ± 0.05 WT 0.27 ± 0.09 P157 0.39 P169 0.21P158 0.62 E171 0.12 G161 0.09 D173 0.46 S163 0.36 M175 0.15 S167 0.15T182 0.52 G170 0.13 G183 0.40 P171 0.12 L184 0.20 S172 0.11 E185 0.43Q173 0.13 V187 0.69 G174 0.15 R188 0.38 R175 0.10 E193 0.85 S181 0.06K194 0.005

TABLE 2(B) <95% Maximal activity FGF21 FGF19 Position IC-50 (μM)Position IC-50 (μM) D159 1.50 L170 2.17 V160 2.69 T172 1.40 S162 2.44S174 3.39 D164 NC D176 NC P165 NC P177 NC L166 2.75 F178 NC M168 NC G1791.93 V169 4.60 L180 NC S176 2.21 V181 1.81 P177 2.57 S189 >2.00 S1780.67 P190 >2.00 Y179 4.16 S191 >2.00 F192 2.97

Additionally, we tested the ability of FGF19 169-194 K194A peptide toantagonize FGF21 activity in Hep3B cells, and found that this peptidewas a superior antagonist compared to the native sequence as seen in theengineered 293T HEK hKLB cells (FIG. 7C). We tested the ability ofFGF21/FGF19 C-terminal peptides to antagonize native FGF19 functionHep3B cells and found them to be effective antagonists, and again theFGF19 169-194 K194A peptide was observed to be a superior antagonist(FIG. 7C).

FGF21 analogs with site-specific alanine mutations predicted by thepeptide antagonist screen were synthesized and assessed for agonistactivity. We chose one mutation which debilitates the antagonism bysubstituting alanine at position 164, and another which preserves theantagonistic function at position 171. Both the site specific alanineFGF21 analogs were tested for their ability to induce Erk1/2phosphorylation in comparison to the native FGF21. We found that theobserved agonistic activity was indeed in complete agreement with theantagonist activity seen by the short peptides. The results validatedthe ability to translate from one molecular format to another.

To confirm that the FGF21 D164A analog was not compromised in itsactivity due to a substantial change in its secondary structure werecorded the CD spectra of the FGF21 D164A and compared it to the nativeFGF21 signature. The CD spectra of the Ala164 FGF21 is comparable tonative FGF21, which implies that the inability to biochemically signalis a function of local and not systemic change to the protein structure.(FIG. 5). FGF19 & 21 share high identity in their C-terminal sequences(FIG. 6). This region based upon analogy with FGF23 is suspected to beof importance in binding to KLB. The results demonstrate that a peptidefragment representing as little as 15% of the full length protein isfully effective in antagonizing native FGF21 in vitro signaling at ˜10×molar concentration. A complete alanine scan of the peptide revealed anumber of sites that sharply differed in antagonistic effectiveness. Themost debilitating alanine mutations occurred in residues that wereidentical among FGF19 and 21, and a specific example is position 164(FIG. 4). These results define the precise positions that constitute thehigh affinity interactions of FGF21 with its co-receptor KLB, andprovide a basis for optimizing protein agonism through analysis ofpeptide-based antagonism (FIG. 6; Table 1).

Positions 8, 9 and 12 were found to be critical residues, whereaspositions 3, 4, 6, 10, 13, 20, 21 and 23 were found to be positionsimpacting activity. The low activity associated with peptides comprisingthese specific amino acid substitutions (when mutated to alanine)identifies these amino acids as part of the putative binding domain forKLB. Eleven of the twenty-five residues within this C-terminal peptidedemonstrated more than a 10× reduction in potency when mutated toalanine.

Example 2

Further modifications to the FGF19 and FGF21 C-terminal sequences.

The initial alanine scan of the FGF19 169-194 K194A peptide fragmentrevealed that substitution of the C-terminal amino acid with an alanine(K194A for FGF19 and S181A for FGF21) significantly enhanced theantagonist properties of the peptide (see Table 2(A)). Furthermore, thecreation of a full length FGF19 analog comprising the FGF19 169-194K194A fragment demonstrated that the FGF29 analog was indeed an improvedanalog in the engineered 293T HEK hKLB cells (FIG. 7C).

To assess the activity of the best antagonist peptide as its agonistcounterpart, we generated a chimeric analog which had the core of FGF211-156 and an extension of the FGF19 169-194 K194A peptide, and evaluatedits activity. We found this to be approximately 5-fold more potent thannative FGF21 (FIG. 7C). This fortifies our previous observations thatthere is a firm correlation between the KLB-binding ability of theC-terminal peptides in isolation and their corresponding function asagonists.

Example 3

Further Mutational Analysis

The results of a D-amino acid scan of the FGF21 C-terminal 25 amino acidpeptide (SEQ ID NO: 191) are presented in Table 3. The activity of eachpeptide (all having the primary sequence of SEQ ID NO: 191) wasdetermined using the assay described in Example 1. Stepwise D-isomermutations within the 25-terminal amino acids of FGF21 (SEQ ID NO: 191)largely mimicked the results of the alanine scan. Two mutations at S11and R19, however significantly increased antagonistic potency of thepeptide over the native terminus by 5- and 2-fold respectively.

TABLE 3 D11 and D19 increased the potency of C25 peptide. Sample IDSequences IC50 (nM) % Activity vs C25 C25 PPDVG SSDPL SMVGP SQGRS PSYAS  283.48 ± 135.61 100.000 (SEQ ID NO: 191) d1pPDVG SSDPL SMVGP SQGRS PSYAS   876.53 ± 537.33  54.16 ± 24.05 d2PpDVG SSDPL SMVGP SQGRS PSYAS  4718.00 ± 2988.67   7.61 ± 1.78 d3PPdVG SSDPL SMVGP SQGRS PSYAS  6101.50 ± 6479.22  15.63 ± 20.51 d4PPDvG SSDPL SMVGP SQGRS PSYAS Ambiguous Ambiguous d6PPDVG sSDPL SMVGP SQGRS PSYAS  1981.85 ± 2045.17  32.49 ± 42.20 d7PPDVG SsDPL SMVGP SQGRS PSYAS Ambiguous Ambiguous d8PPDVG SSdPL SMVGP SQGRS PSYAS Ambiguous Ambiguous d9PPDVG SSDpL SMVGP SQGRS PSYAS Ambiguous Ambiguous d10PPDVG SSDPI SMVGP SQGRS PSYAS Ambiguous Ambiguous d11PPDVG SSDPL sMVGP SQGRS PSYAS    57.32 ± 31.13 596.16 ± 211.38 d12PPDVG SSDPL SmVGP SQGRS PSYAS Ambiguous Ambiguous d13PPDVG SSDPL SMvGP SQGRS PSYAS Ambiguous Ambiguous d15PPDVG SSDPL SMVGp SQGRS PSYAS   374.95 ± 188.36  66.84 ± 34.79 d16PPDVG SSDPL SMVGP sQGRS PSYAS   530.58 ± 289.19  45.98 ± 16.23 d17PPDVG SSDPL SMVGP SqGRS PSYAS   262.48 ± 143.30  90.34 ± 24.24 d19PPDVG SSDPL SMVGP SQGrS PSYAS   151.78 ± 151.66 270.70 ± 136.83 d20PPDVG SSDPL SMVGP SQGRs PSYAS 33700.00 ± 44510.18   2.30 ± 1.83 d21PPDVG SSDPL SMVGP SQGRS pSYAS  7799.67 ± 1501.53   3.80 ± 0.87 d22PPDVG SSDPL SMVGP SQGRS PsYAS Ambiguous Ambiguous d23PPDVG SSDPL SMVGP SQGRS PSyAS Ambiguous Ambiguous d24PPDVG SSDPL SMVGP SQGRS PSYaS  1133.67 ± 435.35  32.29 ± 13.52 d25PPDVG SSDPL SMVGP SQGRS PSYAs   263.50 ± 120.11 118.62 ± 38.73

Combining the mutations that were identified by the alanine and D-isomerscan into a single peptide resulted in antagonists that had elevatedpotency compared to the native C-terminal peptides (see Table 4;lowercase letters identifying amino acids in D-isomer conformation).

TABLE 4 Double mutations of FGF21 and 19 have additive affectsincreasing potency ~10 fold over native peptide. Peptide SequenceIC50 (nM) FGF21 D11 PPDVG SSDPL sMVGP SQGRS PSYAS   19.9 ± 5.24(SEQ ID NO: 185) FGF21 D19 PPDVG SSDPL SMVGP SQGrS PSYAS  52.55 ± 10.82(SEQ ID NO: 186) FGF21 D11D19 PPDVG SSDPL sMVGP SQGrS PSYAS  14.13 ±3.87 (SEQ ID NO: 187) FGF19 A26 LETDS MDPFG LVTGL EAVRS PSFEA  17.89 ±4.57 (SEQ ID NO: 188) FGF19 S10 LETDS MDPFs LVTGL EAVRS PSFES 194.34 ±81.39 (SEQ ID NO: 189) FGF19 S10A26 LETDS MDPFs LVTGL EAVRS PSFEA 10.92 ± 6.33 (SEQ ID NO: 190) FGF21 C25 PPDVG SSDPL SMVGP SQGRS PSYAS182.34 ± 56.35 (SEQ ID NO: 191)

Example 4

FGF19/FGF21 Chimeric Peptides

Chimeric peptides we prepared and tested for their ability to antagonizenative FGF21 signaling using the method of Example 1. The results ofthese experiments are provided in Table 5. In summary, the resultsindicate that maximal activity is obtained in the 25mer C-terminalpeptide that comprises FGF19 amino acids at positions 6-13 with anon-charged amino acid (e.g., alanine) at position 25. Moreparticularly, FGF21 based C-terminal peptide fragments having a terminalalanine at position 25 and the amino acids 6-13 of FGF19 retained theenhanced potency of native FGF19 with the terminal alanine.

TABLE 5 Peptide Sequence IC50 FGF21 PPDVGSSDPLSMVGPSQGRSPSYAS 115.9 ± 4C25 (SEQ ID NO: 177) FGF19 PLETDSMDPFGLVTGLEAVRSPSFEA  19.1 ± 1.5 A25(SEQ ID NO: 178) 0430 PLETDSMDPFGLVGPSQGRSPSFEA  15.9 ± 1.1(SEQ ID NO: 179) 0431 PPDVGSMDPFGLVGPSQGRSPSFEA   4.1 ± 0.1(SEQ ID NO: 180) 0432 PLETDSSDPLSMVGPSQGRSPSFEA 119.8 ± 6.3(SEQ ID NO: 181) 0433 PPDVGSMDPFGLVTGLEAVRSPSYAA  10.7 ± 0.7(SEQ ID NO: 182) 0434 PLETDSSDPLSMVTGLEAVRSPSYAA 263.6 ± 78.8(SEQ ID NO: 183) 0435 PPDVGSSDPLSMVTGLEAVRSPSFEA  65.1 ± 4.8(SEQ ID NO: SEQ ID NO: 184)

Modifying FGF21 by substituting the native C-terminal 25 amino acids ofFGF21 with the potent FGF19 A26 antagonistic 25mer peptide significantlyincreases agonism at both human and mouse KLB (See FIGS. 11A and 11B). Afusion of the N-terminus of FGF21 and highly potent C-terminal peptideFGF19 A26 was synthesized (SEQ ID NO: 192). This fusion, called 0268-1A,was confirmed to have enhanced potency (1.82±0.37 & 2.15±0.54) comparedto both FGF21 (8.42±4.1 & 3.96±1.51) and 19 (65.78±58.19 & 25.67±21.32)in cells that overexpressed both human (FIG. 11A) and mouse (FIG. 11B)KLB. Further modification of 0278-1A, by adding six mutations based onpublished data (A31C, G43C, L98D, L100K, N121D, D127K), produced V20278-1A (SEQ ID NO: 193). The peptide of V2 0278-1A having the 6 aminoacid substitutions in the N-terminus 0278-1A exhibited increased potencyrelative to 0278-1A (See FIG. 12). V2-0278-1A displays ˜2-3× higherpotency (0.35±0.12 nM) versus 0278-1A (1.13±0.28) in cellsoverexpressing human KLB (B).

Additional peptides derived from SEQ ID NO: 180 were investigated fortheir activity as antagonists of FGF21 receptor activity. Table 6provides the results of further derivatives of SEQ ID NO: 180 includingamino acid substitutions at positions 5, 7 and 15. All tested peptideshad similar activities as the peptide of SEQ ID NO: 180.

TABLE 6 Peptide Sequence IC50 0431 PPDVGSMDPFGLVGPSQGRSPSFEA 7.1 ± 3(SEQ ID NO: 180) 0470 PPDVG SMDPF GLVGR SQGRS PSFEA 4.9 ± 1.1(SEQ ID NO: 237) 0480 PPDVF SMDPF GLVGP SQGRS PSFEA 2.6 ± 0.5(SEQ ID NO: 238) 0481 PPDVL SMDPF GLVGP SQGRS PSFEA 2.6 ± 0.8(SEQ ID NO: 239) 0482 PPDVS SMDPF GLVGP SQGRS PSFEA 3.0 ± 1.3(SEQ ID NO: 240) 0486 PPDVG SSDPF GLVGP SQGRS PSFEA 4.7 ± 1.6SEQ ID NO: 2411

A fusion of the N-terminus of FGF21 (modified to comprise thesubstitutions A31C, G43C, L98D, L100K, N121D, and D127K) and highlypotent C-terminal peptides of Table 6 produced the following compounds:

(″FGF21 431A″; SEQ ID NO: 247)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSMDPFGLVGPSQGRSPSFEA; (″FGF21 470A″; SEQ ID NO: 248)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVG SMDPF GLVGR SQGRS PSFEA;(″FGF21 480A″; SEQ ID NO: 249)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVF SMDPF GLVGP SQGRS PSFEA;(″FGF21 481A″; SEQ ID NO: 250)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVL SMDPF GLVGP SQGRS PSFEA;(″FGF21 482A″; SEQ ID NO: 251)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVS SMDPF GLVGP SQGRS PSFEA; and(″FGF21 486A″; SEQ ID NO: 252)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTECHLEIREDGTVGCAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREDLKEDGYNVYQSEAHGLPLHLPGDKSPHRKPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPF GLVGP SQGRS PSFEA.Each of these FGF21 analogs were tested for activity at the FGF21receptor using the cell based assay disclosed in Example 1. Thepolypeptides of SEQ ID NOs 247-252, along with analog of those peptideswhere the C-terminal alanine is replace with a lysine were tested alongwith the native FGF21 polypeptide for activity at the FGF21 receptor.Each compound was tested in triplicate and the average IC50 wasdetermined. The results are indicated below:

FGF21 5.11 ± 0.55 FGF21 480K 1.62 ± 0.54 FGF21 480A 0.43 ± 0.04 FGF216.35 ± 3   FGF21 470K 6.14 ± 1.93 FGF21 470A 0.61 ± 0.06 FGF21 5.39 ±3.19 FGF21 431K  4.3 ± 0.07 FGF21 431A 0.51 ± 0.21 FGF21 2.67 ± 0.9 FGF21 481K 0.56 ± 0.06 FGF21 481A 0.19 ± 0.08 FGF21 4.24 ± 1.07 FGF21482K 1.81 ± 0.18 FGF21 482A 0.22 ± 0.03 FGF21 2.03 ± 0.86 FGF21 486K3.16 ± 0.56 FGF21 486A 0.21 ± 0.03

Example 5

In Vivo Administration of FGF Analogs to Mice

Animals.

C57Bl/6 mice were obtained from Jackson Laboratories and fed adiabetogenic diet from Research Diets: a high-sucrose diet with 58% kcalfrom fat. Mice were group-housed on a 12:12-h light-dark cycle at 22° C.with free access to food and water. All studies were approved by andperformed according to the guidelines of the Institutional Animal Careand Use Committee of the University of Cincinnati. All mice were treatedby daily subcutaneous injections delivered in physiologically bufferedsaline at a dose of 0.3 or 1 mg/kg. Animals were weighed and foodconsumption was measured each day.

Statistical Analyses.

Unless indicated otherwise, all statistical analyses were performedusing GraphPad Prism. The analysis of the results obtained in the invivo experiments was performed using one-way ANOVAs followed by Tukeypost hoc tests. P values lower than 0.05 were considered significant.The results are presented as means±s.e.m. of 7-8 replicates per group.Receptor activation data is ±s.d.

The effect of FGF analog V2-0278-1A on mice was investigated byadministering either vehicle, FGF21 (at 0.3 mg/kg or 1.0 mg/kg) orV2-0278-1A (at 0.3 mg/kg or 1.0 mg/kg) and monitoring weight over thecourse of 6 days of treatment. FIGS. 13A and 13B demonstrate enhancedweight loss in mice receiving FGF analog V2-0278-1A relative to nativeFGF21. FIG. 13C demonstrates that mice receiving FGF analog V2-0278-1Ahad a reduced food intake relative to native FGF21.

1. A peptide exhibiting antagonist activity against FGF21 binding toKlotho β, said peptide comprising an amino acid sequence ofX₁X₂X₃X₄X₅SX₇DPX₁₀X₁₁X₁₂VX₁₄GX₁₆X₁₇X₁₈X₁₉RSPSX₂₄X₂₅X₂₆ (SEQ ID NO: 235),wherein X₁ is Pro or absent; X₂ is Pro or Leu; X₃ is Asp or Glu; X₄ isVal or Thr; X₅ is Gly, Asp, Phe, Leu or Ser; X₇ is Ser or Met; X₁₀ isLeu or Phe; X₁₁ is Ser or Gly; X₁₂ is Met or Leu; X₁₄ is absent or Thr;X₁₆ is Pro, Leu, Arg, Glu, or Gly; X₁₇ is Ser or Glu; X₁₈ is Gln or Ala;X₁₉ is Gly or Val; X₂₄ is Tyr or Phe; X₂₅ is Ala or Glu; and X₂₆ is Ala.2. The peptide of claim 1 wherein said peptide comprises a sequenceselected from the group consisting of I) LETDSMDPFGLVTGLEAVRSPSFEA (SEQID NO: 188), PPDVGSSDPLSMVGPSQGRSPSYAA (SEQ ID NO: 191),PPDVGSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 180), PLETDSMDPFGLVGPSQGRSPSFEA(SEQ ID NO: 179), PDVGSMDPFGLVTGLEAVRSPSYAA (SEQ ID NO: 234) PPDVGSMDPFGLVGR SQGRS PSFEA (SEQ ID NO: 237), PPDVF SMDPF GLVGP SQGRS PSFEA (SEQID NO: 238), PPDVL SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 239), PPDVS SMDPFGLVGP SQGRS PSFEA (SEQ ID NO: 240), and PPDVG SSDPF GLVGP SQGRS PSFEA(SEQ ID NO: 241), or II) a peptide that differs from SEQ ID NO: 188, SEQID NO: 191, SEQ ID NO: 180, SEQ ID NO: 179, SEQ ID NO: 234, SEQ ID NO:237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240 or SEQ ID NO: 241 byone to two amino acid substitutions selected from positions 1, 2, 5, 7,11, 14, 15, 16, 17, 18 and
 19. 3. The peptide of claim 2 wherein saidpeptide is fused to the carboxy terminus of a polypeptide selected fromthe group consisting of SEQ ID NO: 194, SEQ ID NO: 195 and SEQ ID NO:196 or a peptide that differs from SEQ ID NO: 194, SEQ ID NO: 195 or SEQID NO: 196 by 1 to 3 amino acid substitutions.
 4. (canceled)
 5. Amodified FGF21 peptide wherein said modified peptide differs from SEQ IDNO: 173 by a substitution of the C-terminal amino acid with alanine; andone or more of the following modifications: i) one or more substitutionsselected from the group consisting of G161L, G161F, G161S, S163M, L166F,S167G, M168L, P171R, Y179F, and A180E (based on the numbering of themature FGF21 peptide of SEQ ID NO: 173); or ii) one or moresubstitutions selected from the group consisting of A31C, G43C, L98D,L100K, N121D, and D127K; or iii) substitution of the native amino acidat position 167 and/or 175 with the corresponding D-isomer of saidnative amino acid; or iv) substitution of the C-terminal 25 amino acidsof SEQ ID NO: 173 with the 25 amino acid sequence of SEQ ID NO: 188; orv) any combination of i) and ii), or any combination of i), ii) andiii), or any combination of ii) and iv). 6-7. (canceled)
 8. The modifiedFGF21 peptide of claim 5 wherein said modified peptide differs from SEQID NO: 173 by each of the following substitutions: S163M, L166F, S167Gand M168L, or said modified peptide differs from SEQ ID NO: 173 by eachof the following substitutions: S163M, L166F, S167G, M168L, Y179F, andA180E.
 9. (canceled)
 10. The modified FGF21 peptide of claim 8 furthercomprising the substitution of P171R.
 11. The modified FGF21 peptide ofclaim 5 wherein the peptide consists of a peptide selected from thegroup consisting of i) SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 247,SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251 and SEQID NO:252 or ii) SEQ ID NO: 192, SEQ ID NO: 206, SEQ ID NO: 207, SEQ IDNO: 208 and SEQ ID NO: 209, or iii) a modified sequence of SEQ ID NO:192, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208 and SEQ ID NO: 209,wherein SEQ ID NO: 192, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208or SEQ ID NO: 209 are modified to comprise one or more substitutionsselected from the group consisting of A31C, G43C, L98D, L100K, N121D,and D127K. 12-14. (canceled)
 15. A pharmaceutical composition comprisinga modified FGF21 peptide of claim 5 and a pharmaceutically acceptablecarrier, diluent, or excipient.
 16. A method of reducing weight gain orinducing weight loss in a patient in need thereof, comprisingadministering to said patient in need thereof a pharmaceuticalcomposition of claim 15 in an amount effective to reduce weight gain orinduce weight loss.
 17. A method of treating diabetes, comprisingadministering to a patient in need thereof a pharmaceutical compositionof claim 15 in an amount effective to lower blood glucose levels.
 18. Apeptide conjugate comprising the general formula:Q-L-Y wherein Q is a bioactive peptide selected from the groupconsisting of an insulin peptide, a glucagon peptide, FGF1, FGF2, and anuclear hormone; Y is a peptide comprising a sequence selected from thegroup consisting of LETDSMDPFGLVTGLEAVRSPSFEA (SEQ ID NO: 188),PPDVGSSDPLSMVGPSQGRSPSYAA (SEQ ID NO: 191), PPDVGSMDPFGLVGPSQGRSPSFEA(SEQ ID NO: 180), PLETDSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 179),PDVGSMDPFGLVTGLEAVRSPSYAA (SEQ ID NO: 234) PPDVG SMDPF GLVGR SQGRS PSFEA(SEQ ID NO: 237), PPDVF SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 238), PPDVLSMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 239), PPDVS SMDPF GLVGP SQGRS PSFEA(SEQ ID NO: 240), and PPDVG SSDPF GLVGP SQGRS PSFEA (SEQ ID NO: 241);and L is a linking group or a bond.
 19. The conjugate of claim 18wherein i) Q is a glucagon peptide comprising a sequence from the groupconsisting of HX₁QGTFTSDKSKYLDX₂RAAQDFVQWLMDT (SEQ ID NO: 202),(SEQ ID NO: 197) X₃AQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS,(SEQ ID NO: 198) X₄AQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS,(SEQ ID NO: 199) X₅AQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS,(SEQ ID NO: 200) X₆AQGTFTSDKSKYLDERAAQDFVQWLLDAGPSSGAPPPS and(SEQ ID NO: 201) X₇AQGTFTSDKSKYLDERAAQDFVQWLLEAGPSSGAPPPS,

 wherein X₁ and X₂ are both Aib; X₃ is Acetyl D-Tyr; X₄ is Acetyl D-His;X₅ is Acetyl D-thio Ala, and X₆ and X₇ are both acetyl-D-Tyr, or ii) Qis selected from the group consisting of estradiol and derivativesthereof, estrone and derivatives thereof, testosterone and derivativesthereof, and cortisol and derivatives thereof; or iii) Q is a compoundhaving the general structure

wherein R₁₅ is C₁-C₄ alkyl, —CH₂(pyridazinone), —CH₂(OH)(phenyl)F,—CH(OH)CH₃, halo or H; R₂₀ is halo, CH₃ or H; R₂₁ is halo, CH₃ or H; R₂₂is H, OH, halo, —CH₂(OH)(C₆ aryl)F, or C₁-C₄ alkyl; and R₂₃ is—CH₂CH(NH₂)COOH, —OCH₂COOH, —NHC(O)COOH, —CH₂COOH —NHC(O)CH₂COOH,—CH₂CH₂COOH, or —OCH₂PO₃ ²⁻, or iv) Q is a compound having the generalstructure

wherein R₁₅ is C₁-C₄ alkyl, —CH(OH)CH₃, I or H R₂₀ is I, Br, CH₃ or H;R₂₁ is I, Br, CH₃ or H; R₂₂ is H, OH, I, or C₁-C₄ alkyl; and R₂₃ is—CH₂CH(NH₂)COOH, —OCH₂COOH, —NHC(O)COOH, —CH₂COOH, —NHC(O)CH₂COOH,—CH₂CH₂COOH, or —OCH₂PO₃ ²⁻, or v) Q is a compound of the generalstructure of Formula I:

wherein R₂₀, R₂₁ and R₂₂ are independently selected from the groupconsisting of H, OH, halo and C₁-C₄ alkyl; and R₁₅ is halo or H, or vi)Q is selected from the group consisting of thyroxine T4(3,5,3′,5′-tetra-iodothyronine), and 3,5,3′-triiodo L-thyronine; or vii)Q is a ligand that activates the peroxisome proliferator-activatedreceptors (PPAR) when in an unbound state. 20-25. (canceled)
 26. Theconjugate of claim 18 wherein (Q) is an insulin peptide comprising an Achain and a B chain wherein i) said A chain comprises a sequenceGIVX₄X₅CCX₈X₉X₁₀CX₁₂LX₁₄X₁₅LX₁₇X₁₈YCX₂₁-R₅₃ (SEQ ID NO: 19), and said Bchain comprises a sequence R₆₂-X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGX₄₁X₄₂GFX₄₅(SEQ ID NO: 20), wherein X₄ is glutamic acid or aspartic acid; X₅ isglutamine or glutamic acid X₈ is histidine, threonine or phenylalanine;X₉ is serine, arginine, lysine, ornithine or alanine; X₁₀ is isoleucineor serine; X₁₂ is serine or aspartic acid; X₁₄ is tyrosine, arginine,lysine, ornithine or alanine; X₁₅ is glutamine, glutamic acid, arginine,alanine, lysine, ornithine or leucine; X₁₇ is glutamic acid, asparticacid, asparagine, lysine, ornithine or glutamine; X₁₈ is methionine,asparagine, glutamine, aspartic acid, glutamic acid or threonine; X₂₁ isselected from the group consisting of alanine, glycine, serine, valine,threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine,aspartic acid, histidine, tryptophan, tyrosine, and methionine; X₂₅ ishistidine or threonine; X₂₉ is selected from the group consisting ofalanine, glycine and serine; X₃₀ is selected from the group consistingof histidine, aspartic acid, glutamic acid, homocysteic acid and cysteicacid; X₃₃ is selected from the group consisting of aspartic acid andglutamic acid; X₃₄ is selected from the group consisting of alanine andthreonine; X₄₁ is selected from the group consisting of glutamic acid,aspartic acid or asparagine; X₄₂ is selected from the group consistingof alanine, ornithine, lysine and arginine; X₄₅ is tyrosine orphenylalanine; R₆₂ is selected from the group consisting of AYRPSE (SEQID NO: 14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptideglycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine,a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine,glutamine, glutamic acid and an N-terminal amine; and R₅₃ is COOH orCONH₂, or ii) said A chain comprises the sequenceGIVEQCCX₈X₉ICSLYQLENYCX₂₁-R₅₃ (SEQ ID NO: 73) said B chain comprises thesequence R₆₂-X₂₅LCGX₂₉X₃₀LVX₃₃X₃₄LYLVCGX₄₁X₄₂GFX₄₅ (SEQ ID NO: 20),wherein X₈ is histidine or threonine; X₉ is serine, lysine, or alanine;X₂₁ is alanine, glycine or asparagine; X₂₅ is histidine or threonine;X₂₉ is selected from the group consisting of alanine, glycine andserine; X₃₀ is selected from the group consisting of histidine, asparticacid, glutamic acid, homocysteic acid and cysteic acid; X₃₃ is selectedfrom the group consisting of aspartic acid and glutamic acid; X₃₄ isselected from the group consisting of alanine and threonine; X₄₁ isselected from the group consisting of glutamic acid, aspartic acid orasparagine; X₄₂ is selected from the group consisting of alanine,ornithine, lysine and arginine; X₄₅ is tyrosine or phenylalanine; R₆₂ isselected from the group consisting of FVNQ (SEQ ID NO: 12), a tripeptidevaline-asparagine-glutamine, a dipeptide asparagine-glutamine, glutamineand an N-terminal amine; and R₅₃ is COOH or CONH₂, or iii) said A chaincomprises a sequence GIVDECCX₈X₉SCDLRRLEMX₁₉CX₂₁-R₅₃ (SEQ ID NO: 74) andsaid B chain comprises a sequence R₆₂-X₂₅LCGAX₃₀LVDALYLVCGDX₄₂GFY (SEQID NO: 75), wherein X₈ is phenylalanine or histidine; X₉ is arginine,ornithine or alanine; X₁₉ is tyrosine, 4-methoxy-phenylalanine or4-amino-phenylalanine; X₂₁ is alanine or asparagine; X₂₅ is histidine orthreonine; X₃₀ is selected from the group consisting of histidine,aspartic acid, glutamic acid, homocysteic acid and cysteic acid; X₄₂ isselected from the group consisting of alanine ornithine and arginine;and R₅₃ is COOH or CONH₂; R₆₂ is selected from the group consisting ofAYRPSE (SEQ ID NO: 14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), atripeptide glycine-proline-glutamic acid, a tripeptidevaline-asparagine-glutamine, a dipeptide proline-glutamic acid, adipeptide asparagine-glutamine, glutamine, glutamic acid and anN-terminal amine; and R₅₃ is COOH or CONH₂, or iv) said A chaincomprises a sequence GIVDECCX₈X₉SCDLRRLEMX₁₉CX₂₁-R₅₃ (SEQ ID NO: 74) andthe B chain sequence comprises the sequenceFVKQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 147), orFVNQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 148), wherein X₈ isphenylalanine or histidine; X₉ is arginine, ornithine or alanine; X₁₉ istyrosine, 4-methoxy-phenylalanine or 4-amino-phenylalanine; X₂₅ isselected from the group consisting of histidine and threonine; and R₆₃is selected from the group consisting of YTX₂₈KT (SEQ ID NO: 149), YTKPT(SEQ ID NO: 150), YTX₂₈K (SEQ ID NO: 152), YTKP (SEQ ID NO: 151), YTPK(SEQ ID NO: 70), YTX₂₈, YT, Y and a bond, wherein X₂₈ is proline,aspartic acid or glutamic acid, or v) said A chain comprises a sequenceGIVDECCX₈X₉SCDLRRLEMX₁₉CX₂₁—R₅₃ (SEQ ID NO: 74) and the B chain sequencecomprises the sequence FVKQX₂₅LCGSHLVEALYLVCGERGFFYTEKT (SEQ ID NO:162), FVNQX₂₅LCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO: 164),FVNQX₂₅LCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 165) orFVNQX₂₅LCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 161) wherein X₈ isphenylalanine or histidine; X₉ is arginine, ornithine or alanine; X₁₉ istyrosine, 4-methoxy-phenylalanine or 4-amino-phenylalanine; X₂₅ isselected from the group consisting of histidine and threonine; or vi)said A chain comprises a sequence GIVEQCCTSICSLYQLENYCN-R₅₃ (SEQ IDNO: 1) and said B chain comprises a sequenceFVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2), wherein R₅₃ is COOH orCONH₂. 27-31. (canceled)
 32. The conjugate of claim 26, wherein L isstable in vivo, hydrolyzable in vivo, or metastable in vivo.
 33. Theconjugate of claim 32, wherein L comprises an ether moiety, or an amidemoiety, an ester moiety, an acid-labile moiety, a reduction-labilemoiety, an enzyme-labile moiety, a hydrazone moiety, a disulfide moiety,or a cathepsin-cleavable moiety.
 34. The conjugate of claim 18, furthercomprising an acyl group or alkyl group covalently linked to an aminoacid side chain of said conjugate.
 35. A pharmaceutical compositioncomprising a conjugate of claim 18, or pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.
 36. The peptide ofclaim 5 for use in treating diabetes.
 37. The peptide of claim 5 for usein reducing weight gain or inducing weight loss in a patient in needthereof.